Vaccines for broad spectrum protection against diseases caused by Neisseria meningitidis

ABSTRACT

The present invention generally provides methods and vaccines for the prevention of diseases caused by  Neisseria meningitidis  bacteria, particularly serogroup B strains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of earlier-filed U.S. provisionalapplication Ser. No. 60/221,495, filed Jul. 27, 2000, which applicationis incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants no. AI46464and AI45642 awarded by the National Institute of Allergy and InfectiousDiseases, and the National Institute of Health. The government may havecertain rights in this invention.

FIELD OF THE INVENTION

This invention relates to broad-spectrum vaccines for the prevention ofdiseases caused by Neisseria meningitidis, especially serogroup B.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is a Gram-negative bacteria which colonizes thehuman upper respiratory tract and is responsible for worldwide sporadicand cyclical epidemic outbreaks of, most notably, meningitis and sepsis.The attack and morbidity rates are highest in children under 2 years ofage.

Like other Gram negative bacteria, Neisseria meningitidis typicallypossess a cytoplasmic membrane, a peptidoglycan layer, an outer membranewhich together with the capsular polysaccharide constitute the bacterialwall, and pili which project into the outside environment. These surfacestructures mediate infection and interact with the host immune system.For example, a first step in infection with Neisseria is adherence totarget cells, which is thought to be mediated by the pili and, possibly,other adhesins such as Opc. Protein, phospholipid and polysaccharidecomponents of the outer membrane have been reported to elicit an immuneresponse.

Neisseria meningitidis spp. can be divided into serologic groups, typesand subtypes on the basis of reactions with polyclonal (Frasch, C. E.and Chapman, 1973, J. Infect. Dis. 127: 149-154) or monoclonalantibodies (Hussein, A., MONOCLONAL ANTIBODIES AND N. MENINGITIDIS.Proefschrift. Utrecht, Nederland, 1988) that interact with differentsurface antigens. Serogrouping is based on immunologically detectablevariations in the capsular polysaccharide. About 12 serogroups areknown: A, B, C, X, Y, Z, 29-E, W-135, H, I, K and L (Ashton, F. E. etal., 1938, J. Clin. Microbiol. 17: 722-727; Branham, S. E., 1956, Can.J. Microbiol. 2: 175-188; Evans, A. C., 1920, Lab. Bull. 1245: 43-87;Shao-Qing, et al., 1972, J. Biol. Stand. 9: 307-315; Slaterus, K. W.,1961, Ant. v. Leeuwenhoek, J. Microbiol. Serol. 29: 265-271). Currently,serogroup B (MenB) is responsible for about half to 80% of reportedinvasive Neisseria meningitidis diseases.

Serotyping is based on monoclonal antibody defined antigenic differencesin an outer membrane protein called Porin B (PorB). Antibodies definingabout 21 serotypes are currently known (Sacchi et al., 1998, Clin. Diag.Lab. Immunol. 5:348). Serosubtyping is based on antibody definedantigenic variations on an outer membrane protein called Porin A (PorA).Antibodies defining about 18 serosubtypes are currently known.Serosubtyping is especially important in Neisseria meningitidis strainswhere immunity may be serosubtype specific. Most variability betweenPorA proteins occurs in two (loops I and IV) of eight putative, surfaceexposed loops. The variable loops I and IV have been designated VR1 andVR2, respectively. Since more PorA VR1 and VR2 sequence variants existthat have not been defined by specific antibodies, an alternativenomenclature based on VR typing of amino acid sequence deduced from DNAsequencing has been proposed (Sacchi et al., 2000, J. Infect. Dis.182:1169; see also the Multi Locus Sequence Typing web site).Lipopolysaccharides can also be used as typing antigens, giving rise toso-called immunotypes: L1, L2, etc.

Neisseria meningitidis also may be divided into clonal groups orsubgroups, using various techniques that directly or indirectlycharacterize the bacterial genome. These techniques include multilocusenzyme electrophoresis (MLEE), based on electrophoretic mobilityvariation of an enzyme, which reflects the underlying polymorphisms at aparticular genetic locus. By characterizing the variants of a number ofsuch proteins, genetic “distance” between two strains can be inferredfrom the proportion of mismatches. Similarly, clonality between twoisolates can be inferred if the two have identical patterns ofelectrophoretic variants at number of loci. More recently, multilocussequence typing (MLST) has superseded MLEE as the method of choice forcharacterizing the microorganisms. Using MLST, the genetic distancebetween two isolates, or clonality is inferred from the proportion ofmismatches in the DNA sequences of 11 housekeeping genes in Neisseriameningitidis strains (Maiden et al., 1998, Proc. Natl. Acad. Sci. USA95:3140).

Given the prevalence and economic importance of invasive Neisseriameningitidis infections, it is not surprising that many attempts havebeen made to develop treatments. Although these infections can betreated with antibiotics, about 10 to 20% of treated patients die, andmany survivors are left with permanent neurologic sequelae, such asamputation, neurosensory hearing loss, and paralysis. Also,microorganisms can develop antibiotic resistance. Thus, prevention withvaccines is a preferable mode to contain the spread of infection.

Because the polysaccharide capsule is one of the outermost structures ofpathogenic Neisseria meningitidis, it has been a primary focus ofattempts to develop vaccines. Different preparations of capsularpolysaccharides have been used to control the outbreaks and epidemics ofthe serogroups A, C, Y and W-135, as mono-, di-, tri- or tetravalentvaccines (Gold et al., 1969-1970, Bull. WHO 45: 272-282; Gotschlich etal., 1969, J. Exp. Meal. 129: 134-136; Hankins, 1982, Proc. Soc. Biol.Med. 169: 54-57; U.S. Pat. No. 6,080,589). However, capsularpolysaccharide vaccines suffer from: poor or no-response topolysaccharide C in children under 2 years of age; thermolability ofpolysaccharide A; difficulties regarding the induction of immunologictolerance after vaccination or re-vaccination with polysaccharide C(Granoff et al., 1998, J. Infect. Dis. 160: 5028-5030; MacDonald et al.,1998, JAMA 280:1685-1689; MacDonald et al., 2000, JAMA 283: 1826-1827).To circumvent these immunologic properties, polysaccharides fromserogroups A and C have been covalently coupled to protein carriers tomake “conjugate” vaccines. In contrast to plain polysaccharide vaccines,these conjugate vaccines are highly immunogenic in infants, uponre-injection elicit boostable increases in serum anticapsular antibodyconcentrations, and prime for the ability to generate memory antibodyresponses to a subsequent injection of plain polysaccharide (Campagne etal. 2000, Pediat. Infect. Dis. J. 19: 144-150; Maclennan et al., 2000,JAMA 283: 2795-2801). Conjugate vaccines with similar properties havebeen highly effective in preventing invasive diseases caused by otherencapsulated bacteria, such as Haemophilus influenzae type b orStreptococcus pneumoniae.

The capsular polysaccharide (PS) of serogroup B Neisseria meningitidisis a very poor immunogen in humans (Wyle et al., 1972, J. Infect. Dis.126: 514-522; Zollinger, et al., 1979, J. Clin. Invest. 63: 836-834;Jennings et al., 1981, J. Immunol. 127: 104-108). Further attempts toimprove the polysaccharide's immunogenicity through conjugation toprotein have been unsuccessful (Jennings et al., 1981, J. Immunol. 127:104-108). To enhance the immunogenicity, the meningococcal serogroup Bcapsule polysaccharide (MenB PS) has been chemically modified(N-propionylated group was substituted for the N-acetyl group of Bpolysaccharide) and coupled covalently to a protein carrier (N-Pr-MenBPS-protein) conjugate. The vaccine induces in mice high titers of IgGantibodies which are bactericidal and protective (this concept isdescribed and claimed in U.S. Pat. No. 4,727,136, issued Feb. 23, 1988to Jennings et al.). This vaccine also is immunogenic in sub-humanprimates, inducing serum antibodies that activate complement-mediatedbacteriolysis (Fusco et al., 1997, J. Infect. Dis. 175: 364-372). Inhumans, such antibodies are known to confer protection againstdeveloping meningococcal disease (Goldschneider et al., 1969, J. Exp.Med. 129:1307). However, a subset of the antibodies induced by thisvaccine have autoantibody activity to unmodified MenB PS (i.e.N-acetyl-MenB PS), Granoff et al., 1998, J. Immunol; 160: 5028-5036,which raise serious safety concerns about the use of this vaccine inhumans. Therefore, investigators have sought alternative approaches todevelop a safe and effective vaccine for prevention of disease caused byserogroup B strains.

Other groups have focused on surface proteins as vaccines. For example,the principal protein component of the pilus, pilin, elicits an immuneresponse; however, so many antigenic variants exist and continue todevelop that vaccines against the pilus protein have not been highlyeffective. See, U.S. Pat. No. 5,597,572. In other examples, vaccineshave focused the highly conserved Neisserial surface protein A (NspA)(see, e.g., PCT Publication No. WO96/29412). Although the gene is highlyconserved and expressed in virtually all strains, both polyclonal andmonoclonal antibodies prepared against recombinant NspA are bactericidaland/or provide protection, against only about 50% of genetically diversestrains (Moe et al. (1999 Infect. Immun. 67: 5664; Moe et al. InfectImmun. 2001 69:3762). These observations suggest that recombinant NspAalone will not provide adequate protection against a broad spectrum ofNeisserial strains.

Still other groups have used membrane preparations to induce immunity.In general, attempts to produce a meningococcal B vaccine based on outermembrane vesicles used repeated immunizations with material preparedfrom a single strain or repeated immunization with a vaccine containingvesicle antigens from multiple strains. When the vaccine containedvesicle antigens from more than strain, the resulting bactericidalantibody titers of infants or children given two or three doses were low(Cartwright K et al, 1999, Vaccine; 17:2612-2619; de Kleinjn E D et al,2000, Vaccine, 18:1456-1466), In these studies, and in a study done incynomolgus monkeys (Rouupe van der Voort E R, 2000, Vaccine,18:1334-1343) there also was evidence of immune interference between theresponses to the different antigen. When repeated immunization withvesicles from a single strain was used, higher antibody titers resultedbut the spectrum of antibody reactivity was limited to only a fewstrains that tended to be serologically similar to each other (Tapperoet al., 1999, JAMA 281:1520; and Rouupe van der Voort E R, 2000,Vaccine, 18:1334-1343). Our experiments in laboratory animal models,which are described below confirmed this latter observation. Antiserafrom control animals given two sequential immunizations of a outermembrane vesicle vaccine prepared at the National Institute of PublicHealth, Oslo, Norway, from a single Neisseria meningitidis serogroup Bstrain, H44/76 (B:15:P1.7,16; “Norwegian vaccine”), reacted by flowcytometry and were bactericidal against only serogroup B strains thatwere of the same serosubtype (i.e. P1.7,16) or strains having an epitopesimilar to the P 1.16 epitope (such as P1.10-4 strains).

Humans are the only known reservoir for Neisseria meningitidis spp.Accordingly, Neisserial species have evolved a wide variety of highlyeffective strategies to evade the human immune system. These includeexpression of a polysaccharide capsule that is cross-reactive with hostpolysialic acid (i.e. serogroup B) and high antigenic mutability for theimmunodominant noncapsular epitopes, i.e. epitopes of antigens that arepresent at the surface in relatively large quantities, are accessible toantibodies, and elicit a strong antibody response.

Prior efforts to develop broad spectrum vaccines have been hampered bythe wide variety of highly effective strategies used by Neisserialspecies to evade the human immune system. Because of these strategies,an immune response to a given strain will often not confer effectiveimmunity against other strains of Neisseria. The present inventionovercomes the disadvantages of prior art approaches to vaccination andelicits protective immunity against a broad spectrum of Neisseriameningitidis strains, notably (but not exclusively) including strainsbelonging to serogroup B.

SUMMARY OF THE INVENTION

The present invention generally provides methods and vaccines for theprevention of diseases caused by Neisseria meningitidis bacteria,particularly serogroup B strains.

In one embodiment, the method of the invention comprises: administeringto a mammal a first preparation of i) outer membrane vesicles (OMV) of afirst Neisseria meningitidis spp., and/or ii) microvesicles (MV)released into a culture medium during culture of a first Neisseriameningitidis spp., said administering of OMV and/or MV being in asufficient amount to immunologically prime and/or elicit an immuneresponse to epitopes present in said first preparation; administering atleast a second preparation of i) OMVs of a second Neisseria meningitidisspp., and/or ii) MVs released into a culture medium during culture of asecond Neisseria meningitidis spp., said administering of OMV and/or MVbeing in a sufficient amount to immunologically prime and/or elicit animmune response to epitopes present in said second preparation; andoptionally, but preferably, administering a third preparation of i) OMVof a third Neisseria meningitidis spp., and/or ii) MV that are releasedinto a culture medium during culture of a third Neisseria meningitidisspp., said administering of OMV and/or MV being in a sufficient amountto elicit an immune response to epitopes present in said thirdpreparation. Administration of the first, second, and (optionally) thirdpreparation results in induction of an immune response to epitopespresent in the preparations, wherein said response confers protectiveimmunity against a disease caused by Neisseria meningitidis spp.

In preferred embodiments, the first, second, and third Neisseria strainsare genetically diverse to one another, e.g., the first strain isgenetically diverse to the second strain, the third strain, or both thesecond and third strain.

In related embodiments, administration of the preparations is serial.Serial administration of the preparations can be conducted in any order.For example, the following orders of administration are within the scopeof the invention (from left to right, with the third administrationbeing optional): OMV-OMV-OMV; OMV-OMV-MV; OMV-MV-MV; MV-MV-MV;MV-MV-OMV; MV-OMV-OMV; OMV-MV-OMV; and MV-OMV-MV. Preferably, the orderof administration is MV-MV-OMV.

In other related embodiments, the preparations are administered as amixture, where the initial administration of the mixture can be followedby one or more additional administrations of the same or differentmixture to serve as boosters.

In one specific embodiment, the invention involves seriallyadministering microvesicles (MV) that bleb naturally during growth ofNeisseria meningitidis and are released in the culture medium (collectedby separating the larger cells from the smaller blebs and then pelletingthe blebs) and/or outer membrane vesicles (OMV, prepared directly fromisolated outer membrane fractions). The OMVs and MVs are prepared from“genetically diverse” strains of Neisseria meningitidis, for example,strains that differ from one another in at least one of serotype orserosubtype, and may be diverse at multiple genetic loci, e.g., differin both serotype and serosubtype, e.g., having different outer membranePorin, PorA and PorB proteins. Further, the OMV and/or MV preparationscan be given sequentially in at least two, and preferably at least threeadministrations (e.g., injections) of OMVs or MVs from geneticallydiverse strains; four, five, six or more administrations are alsocontemplated.

In another specific embodiment, the first Neisseria meningitidis spp. isa member of a first serosubtype; the second Neisseria meningitidis spp.is a member of a second serosubtype, which second subserotype aredifferent from the subserotype of the first Neisseria meningitidis spp,and, where used, the third Neisseria meningitidis spp. is a member of athird serosubtype, which third subserotype is different from thesubserotype of at least the first, and preferably both the first and thesecond, Neisseria meningitidis spp.

In still another specific embodiment, the first Neisseria meningitidisspp. is a member of a first serotype and of a first serosubtype; thesecond Neisseria meningitidis spp. is a member of a second serotype andof a second serosubtype, which second serotype and second subserotypeare different from the serotype and subserotype of the first Neisseriameningitidis spp, and, where used, the third Neisseria meningitidis spp.is a member of a third serotype and of a third serosubtype, which thirdserotype and third subserotype are different from the serotype andsubserotype of at least the first, and preferably both the first and thesecond, Neisseria meningitidis spp.

In one specific embodiment of the invention, a first administration iswith microvesicles (MVs) prepared from a serogroup C strain (e.g. RM1090(C:2a:P1.5,2:L3,7)). The second administration is with MVs prepared froma second strain (e.g. BZ198 (B:NT:P1.4)), and the third administrationis with outer membrane vesicles (OMVs) prepared from a third strain(e.g. Z1092 (A:4,21:P1.10)). Sequential immunization with vesiclesand/or microvesicles prepared from genetically diverse Neisseriameningitidis strains is referred to hereafter as the “CHORI vaccine” or“CHORI antigen.” Immunization with a mixture of the first, second, andthird preparations of the CHOR vaccine is referred to as “CHORI mix”.

In other aspects, the invention features a composition comprising afirst preparation selected from the group consisting of outer membranevesicle (OMV), microvesicles (MV), or both OMV and MV from a first froma first Neisseria meningitidis species; a second preparation selectedfrom the group consisting of outer membrane vesicle (OMV), microvesicles(MV), or both OMV and MV from a second Neisseria meningitidis species,wherein the second Neisseria meningitidis spp is genetically diverse tothe first Neisseria meningitidis species; and a pharmaceuticallyacceptable carrier.

In related embodiments, the composition further comprises a thirdpreparation selected from the group consisting of outer membrane vesicle(OMV), microvesicles (MV), or both OMV and MV from a third Neisseriameningitidis species, wherein the third Neisseria meningitidis speciesis genetically diverse to the first Neisseria meningitidis species. Inspecific embodiments, the first preparation of the composition comprisesMV, the second preparation comprises MV; and the third preparationcomprises OMV. Preferably, the first and second Neisseria meningitidisspecies are genetically diverse in that they differ in at least one ofserotype or serosubtype, and, where included, the third and the firstNeisseria meningitidis species are genetically diverse in that theydiffer in at least one of serotype or serosubtype.

In still other aspects, the invention features a composition comprisingat least one isolated Neisseria meningitidis antigen, the isolatedantigen being present in the composition in an amount effective toelicit an immune response in a mammalian host, and being characterizedas a protein immunoprecipitated with anti-sera produced followingvaccination with the CHORI-vaccine, and having an apparent molecularmass selected from the group consisting of about 80 kDa, about 59.5 kDa,about 40.7 kDa, about 39.6 kDa, about 33 kDa, about 27.9 kDa, and 14.5kDa; and a pharmaceutically acceptable excipient.

In another aspect, the invention features a composition comprising atleast one isolated Neisseria meningitidis antigen, the isolated antigenbeing present in the composition in an amount effective to elicit animmune response in a mammalian host, and being characterized as aprotein detected by Western blot with anti-sera produced followingvaccination of a mammal with the CHORI vaccine, and having an apparentmolecular mass selected from the group consisting of about 53 kDa to 57kDa; about 46-47 kDa, about 33 kDa, about 20 kDa to 21 kDa; and about 18kDa; and a pharmaceutically acceptable excipient.

In another aspect the invention features a composition comprising atleast one isolated Neisseria meningitidis antigen, the isolated antigenbeing present in the composition in an amount effective to elicit animmune response in a mammalian host, wherein the antigen is from aprotein that specifically binds a monoclonal antibody selected from thegroup consisting of 1D9, 4B11, 9B8, and 14C7 (which antibodies aredescribed herein and deposited with the ATCC); and a pharmaceuticallyacceptable excipient.

In preferred embodiments, the compositions having isolated antigenscomprise at least two isolated Neisseria meningitidis antigens.

In related aspects, the invention features methods for eliciting broadspectrum protective immunity against a disease caused by a Neisseriameningitidis species, said method comprising administering to a mammalat least one of the compositions comprising isolated antigens asdescribed above.

Preferably, the antigen compositions (e.g., OMV/MV preparations,isolated protein preparations) may be administered to mammals,especially humans, that are immunologically naive with respect toNeisseria meningitidis (i.e., have not been exposed to antigens fromNeisseria meningitidis, or have not been exposed insufficient amounts toelicit a protective immune response). A specific embodiment of theinvention involves administration to human infants that are about fiveyears old or younger, especially two years old or younger.

In some embodiments of the invention, prior to administration of antigencompositions from Neisseria meningitidis, the individuals may have beenprimed by exposure (through natural infection or administration) to aNeisserial species other than Neisseria meningitidis (or an antigencomposition prepared from a Neisserial species).

Antisera obtained from mice immunized as described above bind to thebacterial cell surface of a group of genetically diverse Neisseriameningitidis serogroup B strains, as determined by flow cytometricdetection of indirect immunofluorescence.

In one example, sera from immunized mice were positive for eleven of 12strains tested. These 11 included 3 meningococcal B strains withrespective PorA and PorB proteins that were heterologous to those of themeningococcal strains used to prepare the immunogens used forvaccination. (By way of contrast, antisera from animals immunized withtwo injections of the above-described “Norwegian OMV vaccine” reacted byflow cytometry with only 5 of 11 strains. All 5 had PorA and/or PorBproteins that were the same or closely related to those in the“Norwegian” OMV vaccine.) The antisera from animals immunized with the“CHORI vaccine” also elicited complement-mediated bacteriolysis in 11 of12 strains, a good predictor of protection against disease in humans(Goldschneider et al, 1969, J. Exp. Med. 129:1307). Antibody binding tothe bacterial cell surface, or complement-mediated bacteriolysis, wasnot inhibited by the presence of excess soluble serogroup Bpolysaccharide, evidence that the protective antibodies were directedagainst non-capsular antigens.

Antisera from mice immunized in a second example using the CHORI vaccinewere bactericidal against 14 of 14 strains tested including eightstrains with serosubtypes that were heterologous from those used in thevaccine preparations. In a third example, antisera prepared from guineapigs immunized with the CHORI vaccine were bactericidal against 9 of 10strains tested including 5 strains with serosubtypes that wereheterologous to those expressed by the vaccine strains. Antisera to theCHORI vaccine prepared in mice in the first example and in guinea pigsin the third example also were highly protective against bacteremia inthe infant rats challenged with serogroup B bacteria. The immunizationprotocol used herein generally induces the immune system to converge onnon-capsular antigens that are common to the strains from which the MVsand OMVs are obtained. The CHORI vaccine elicits antibodies againstmultiple cell surface epitopes, including, PorA, possibly PorB, andconserved proteins such as Neisserial surface protein A (NspA), theclass 4 protein, (reduction modifiable protein, Rmp) and othernoncapsular antigens as yet unidentified.

In general, the vaccines of the present invention that employ sequentialimmunization with antigenic material prepared from different strains(genetically diverse) have the potential to confer protection againstthe majority of Neisseria meningitdis serogroup B strains. This approachalso has broad applicability for vaccination against Neisseriameningitidis strains representative of other serogroups such as A, C, Y,or W-135, and also against other members of the genus Neisseria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 summarizes the results of meningococcal outer membrane vesiclevaccine efficacy trials.

FIG. 2 is a photograph of a 15% SDS-PAGE gel of microvesicle (MV),deoxycholate extracted microvesicle (DOC MV), outermembrane vesicle(OMV), and deoxycholate extracted outermembrane vesicle (DOC OMV)vaccine preparations from meningococcal strains Z1092 (A:4, 21:P1.10),BZ198 (B:NT:P.1.4), and RM1090 (C:2a:P1.2), respectively. Lane 1,molecular mass standards. Lane 2, Z1092 MV. Lane 3, Z1092 DOC MV. Lane4, Z1092 OMV. Lane 5, Z1092 DOC OMV. Lane 6, BZ198 MV. Lane 7, BZ198 DOCMV. Lane 8, BZ198 OMV. Lane 9, BZ198 DOC OMV. Lane 10, RM1090 MV. Lane11, RM1090 DOC MV. Lane 12, RM1090 OMV. Lane 13, RM1090 DOC OMV.

FIG. 3 is a series of graphs showing binding of anti-CHORI vaccine,anti-Norwegian vaccine and control antisera and mAb to live encapsulatedmeningococcal B strains MC58 (B:15:P1.7,16) and S3446(B:19,14:P1.23,14), as determined by indirect fluorescence flowcytometry. All antisera were tested at dilutions of 1:20. The controlmAb is an anti-capsule-specific murine mAB (Granoff et al., 1998, J.Immunol. 160: 5028-5036). The control antisera were pooled sera frommice immunized with proteins from the supernatant culture of E. colistrain BL21 or guinea pigs immunized with the adjuvant, aluminumhydroxide, alone. Note that the strain MC58 has the same serotype andserosubtype as the strain used to prepare OMV for the Norwegian vaccine.The serotype and serosubtype of strain S3446 is heterologous to thestrains used to prepare both vaccines.

FIG. 4 presents data regarding the bacterial cell surface binding ofantisera determined by indirect fluorescence flow cytometry

FIG. 5 presents data illustrating the reactivity of CHORI antiseraagainst N. meningitidis serogroup A and C strains.

FIG. 6 summarizes the results of a bactericidal assay testing anti-CHORIvaccine, anti-rNspA, and anti-Norwegian vaccine antisera againstmeningococcal B strain 2996.

FIG. 7 provides data showing the complement-mediated bactericidalactivity of antisera and antibodies.

FIG. 8 provides data showing the complement-mediated bactericidalactivity of antisera from mice immunized with the indicated vaccines.

FIG. 9 provides data showing the bactericidal activity of antisera fromguinea pigs immunized with the indicated vaccines.

FIG. 10 provides data showing the passive protection in infant ratsagainst meningococcal B strain 8047 bacteremia by antisera andantibodies.

FIG. 11 provides data showing the passive protection in infant ratsagainst meningococcal B strain 8047 bacteremia by guinea pig antisera.

FIG. 12 is a photograph of a silver stained 15% SDS-PAGE gel ofsurface-exposed proteins precipitated by anti-CHORI antigen antiserafrom non-encapsulated meningococcal B strain M7. Lane 1, total proteinfrom M7. Lane 2, proteins precipitated by murine anti-CHORI antisera.Lane 3, proteins precipitated by murine negative control antisera. Thenumbers on the left of the figure indicate apparent molecular mass inkDa.

FIG. 13 is a photograph of a Western blot of a 15% SDS-PAGE gel ofsurface-exposed proteins precipitated by anti-CHORI antigen antiserafrom non-encapsulated meningococcal B strain M7. Lanes 1 and 4, totalprotein from M7. Lanes 2 and 5, proteins precipitated by murineanti-CHORI antigen antisera. Lanes 3 and 6, proteins precipitated bymurine negative control antisera. Anti-CHORI antigen antisera were usedas the primary detecting antibody in lanes 1 to 3 and an anti-PorA mAbMN16C13F4 (Rijksinstituut Voor Volksgezondeid en Mileu, Biltoven, TheNetherlands) that is specific for serosubtype P1.2 was used as theprimary detecting antibody in lanes 4 to 5.

FIG. 14 provides data showing the bacterial surface accessible proteinsprecipitated by pooled antisera from mice sequentially immunized withMenC strain RM1090 MV, MenB strain BZ198 MV, and MenA strain Z1092 OMV.

FIG. 14A provides additional examples of data showing the bacterialsurface accessible proteins precipiated by pooled anitsera from micesequentially immunized with MenC strain RM1090 MV, MenB strain BZ198 MV,and MenA strain Z1092 OMV.

FIG. 15 is a photograph of a Western blot of a 15% SDS-PAGE gel of MV orOMV preparations. Primary detecting antisera is pooled mouseanti-CHORI/CFA vaccine antisera in lanes 1 to 3, pooled mouseanti-CHORI/Al₂(OPO₃)₃ in lanes 4 and 5, and pooled guinea piganti-CHORI/Al₂(OPO₃)₃ in lanes 6 to 8. Lanes 1 and 6, MV proteinsprepared from strain RM 1090. Lanes 2, 4, and 7, MV proteins preparedfrom strain BZ198. Lanes 3, 5, and 8, OMV proteins prepared from strainZ1092. The numbers on the left of the figure indicate apparent molecularmass in kDa.

FIG. 16 provides data showing the apparent molecular masses of proteinsfrom the indicated MV or OMV preparations that are reactive withantisera from mice and guinea pigs that were sequentially immunized withMV from MenC strain RM1090 and MenB strain BZ198, and OMV from MenAstrain Z1092.

FIG. 17 provides data from ELISA showing the absorption of anti-LOSantibodies from pooled antisera obtained from mice and guinea pigssequentially immunized with MV from MenC strain RM1090 and MenB strainBZ198, and OMV from MenA strain Z1092, or three injections of a mixtureof the three vesicle preparations.

FIG. 18 provides data from complement-mediated bactericidal assayshowing that the absorption of anti-LOS antibodies from pooled antiseraobtained from mice and guinea pigs sequentially immunized with MV fromMenC strain RM1090 and MenB strain BZ198, and OMV from MenA strainZ1092, or three injections of a mixture of the three vesiclepreparations does not significantly change the bactericidal activity ofthe antisera against MenB strains that are homologous or heterologous tothe vaccine strains.

FIG. 19 provides data from a whole cell ELISA showing examples of mAbsproduced from mice sequentially immunized with MV from MenC strainRM1090 and MenB strain BZ198, and OMV from MenA strain Z1092. SeveralmAbs are reactive with all meningococcal strains tested and others reactwith a limited subset of strains.

FIG. 20 summarizes the complement-mediated bactericidal activity of mAbsprepared from mice immunized with anti-CHORI antigen and tested againstseveral MenB strains.

FIG. 21 summarizes the meningococcal serotype and serosubtype definingmonoclonal antibodies available from RIVM.

FIG. 22 summarizes the serogroup, serotype, and serosubtype definingmonoclonal antibodies available from NIBSC.

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantigen” includes a plurality of such antigens and reference to “thevesicle” includes reference to one or more vesicles and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

Immunization of infants, older children and adults with meningococcalouter membrane vesicle (OMV) vaccines induces serum bactericidalantibodies, a serological correlate of protection against disease(Goldschneider et al,1969, J. Exp. Med. 129:1307). The efficacy of OMVvaccines for prevention of meningococcal B disease also has beendemonstrated directly in older children and adults in randomized,prospective clinical trials, and in retrospective case-control studies.See, e.g., results summarized in background section and in FIG. 1. Thus,the clinical effectiveness of outer membrane vesicle vaccines is not indispute. Such vaccines are close to licensure for use in Norway in olderchildren and adults, and are in late-stage clinical development forlicensure in other European countries. An OMV vaccine prepared by theFinley Institute in Cuba also is available commercially and has beengiven to millions of children in South America.

The serum bactericidal antibody response to OMV vaccines tends to bestrain specific (Tappero et al., 1999, JAMA 281:1520; and Rouupe van derVoort E R, 2000, Vaccine, 18:1334-1343). PorA is immunodominant, and theimmunity induced is predominantly specific to the strains from which themembrane vesicles were obtained (Tappero et al., 1999, JAMA 281:1520;Martin S L et al, 2000, Vaccine, 18:2476-2481). This limitation isprimarily because of antigenic variability of the PorA protein and isparticularly true in infants who are immunologically naïve (Tappero etal.) with respect to prior exposure to neisserial antigens.

Hence, the present invention involves eliciting an immune response thatis broadly reactive with diverse disease-producing N. meningitidisstrains. The invention circumvents the problem of immunodominance ofantigenically variable domains of PorA in vesicle-or PorA-based vaccinesby focusing the antibody response on common antigens in the vaccinestrains. Importantly, the methods of the invention elicit serumbactericidal antibody, the only proven serologic correlate of protectionin humans (Goldschneider et al. 1969, supra), against strains ofNeisseria expressing serosubtype epitopes that were not used in thevaccine preparations. Further, the method elicits serum bactericidalantibody against strains that are not killed by antibody to a conservedprotein such as Neisserial surface protein A, a candidate meningococcalvaccine (Martin et al., 2000. J. Biotechnol. 83:27-31; Moe et al. (1999Infect. Immun. 67: 5664; Moe et al. Infect Immun. 2001 69:3762). Withoutbeing held to theory, the vaccine and immunization regimen of theinvention provides its unexpected advantages in broad spectrumprotective immunity by eliciting antibodies that are specific for bothconserved and non-conserved antigens.

A. Definitions

The term “protective immunity” means that a vaccine or immunizationschedule that is administered to a mammal induces an immune responsethat prevents, retards the development of, or reduces the severity of adisease that is caused by Neisseria meningitidis, or diminishes oraltogether eliminates the symptoms of the disease.

The phrase “a disease caused by a strain of serogroup B of Neisseriameningitidis” encompasses any clinical symptom or combination ofclinical symptoms that are present in an infection with a member ofserogroup B of Neisseria meningitidis. These symptoms include but arenot limited to: colonization of the upper respiratory tract (e.g. mucosaof the nasopharynx and tonsils) by a pathogenic strain of serogroup B ofNeisseria meningitidis, penetration of the bacteria into the mucosa andthe submucosal vascular bed, septicemia, septic shock, inflammation,haemmorrhagic skin lesions, activation of fibrinolysis and of bloodcoagulation, organ dysfunction such as kidney, lung, and cardiacfailure, adrenal hemorrhaging and muscular infarction, capillaryleakage, edema, peripheral limb ischaemia, respiratory distresssyndrome, pericarditis and meningitis.

The phrase “broad spectrum protective immunity” means that a vaccine orimmunization schedule elicits “protective immunity” against at least oneor more (or against at least two, at least three, at least four, atleast five, against at least eight, or at least against more than eight)strains of Neisseria meningitidis, wherein each of the strains belongsto a different serosubtype as the strains used to prepare the vaccine.

The invention specifically contemplates and encompasses a vaccine orvaccination regimen that confers protection against a disease caused bya member of serogroup B of Neisseria meningitidis and also against otherserogroups, particularly serogroups A, C, Y and W-135.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to an antigen such as apolysaccharide, phospholipid, protein or peptide, refers to a bindingreaction which is based on and/or is probative of the presence of theantigen in a sample which may also include a heterogeneous population ofother molecules. Thus, under designated immunoassay conditions, thespecified antibody or antibodies bind(s) to a particular antigen orantigens in a sample and do not bind in a significant amount to othermolecules present in the sample. Specific binding to an antibody undersuch conditions may require an antibody or antiserum that is selectedfor its specificity for a particular antigen or antigens.

The phrase “in a sufficient amount to elicit an immune response toepitopes present in said preparation” means that there is a detectabledifference between an immune response indicator measured before andafter administration of a particular antigen preparation. Immuneresponse indicators include but are not limited to: antibody titer orspecificity, as detected by an assay such as enzyme-linked immunoassay(ELISA), bactericidal assay, flow cytometry, immunoprecipitation,Ouchter-Lowny immunodiffusion; binding detection assays of, for example,spot, Western blot or antigen arrays; cytotoxicity assays, etc.

A “surface antigen” is an antigen that is present in a surface structureof Neisseria meningitidis (e.g. the outer membrane, inner membrane,periplasmic space, capsule, pili, etc.).

The phrase “genetically diverse” as used in the context of geneticallydiverse strains of Neisseria meningitidis, refers to strains that differfrom one another in the amino acid sequence of at least one, and usuallyat least two, more usually at least three polypeptides, particularlyantigenic polypeptides. Genetic diversity of strains can be accomplishedby selecting strains that differ in at least one or more, preferably atleast two or more, of serogroup, serotype, or serosubtype (e.g., twostrains that differ in at least one of the proteins selected from outermembrane, PorA and PorB proteins, are said to genetically diverse withrespect to one another). Genetic diversity can also be defined by, forexample, multi-locus sequence typing and/or multi-locus enzyme typing(see, e.g., Maiden et al., 1998, Proc. Natl. Acad. Sci. USA 95:3140;Pizza et al. 2000 Science287: 1816), multi-locus enzyme electrophoresis,and other methods known in the art.

“Serogroup” as used herein refers to classification of Neisseriameningitides by virtue of immunologically detectable variations in thecapsular polysaccharide. About 12 serogroups are known: A, B, C, X, Y,Z, 29-E, W-135, H, I, K and L. Any one serogroup can encompass multipleserotypes and multiple serosubtypes.

“Serotype” as used herein refers to classification of Neisseriameningitides strains based on monoclonal antibody defined antigenicdifferences in the outer membrane protein Porin B. A single serotype canbe found in multiple serogroups and multiple serosubtypes.

“Serosubtype” as used herein refers classification of Neisseriameningitides strains based on antibody defined antigenic variations onan outer membrane protein called Porin A, or upon VR typing of aminoacid sequences deduced from DNA sequencing (Sacchi et al., 2000, J.Infect. Dis. 182:1169; see also the Multi Locus Sequence Typing website). Most variability between PorA proteins occurs in two (loops I andIV) of eight putative, surface exposed loops. The variable loops I andIV have been designated VR1 and VR2, respectively. A single serosubtypecan be found in multiple serogroups and multiple serotypes.

“Enriched” means that an antigen in an antigen composition ismanipulated by an experimentalist or a clinician so that it is presentin at least a three-fold greater concentration by total weight,preferably at least 10-fold greater concentration, more preferably atleast 100-fold greater concentration, and most preferably at least1,000-fold greater concentration than the concentration of that antigenin the strain from which the antigen composition was obtained. Thus, ifthe concentration of a particular antigen is 1 microgram per gram oftotal bacterial preparation (or of total bacterial protein), an enrichedpreparation would contain at least 3 micrograms per gram of totalbacterial preparation (or of total bacterial protein).

The term “immunologically naive with respect to Neisseria meningitidis”denotes an individual (e.g., a mammal such as a human patient) that hasnever been exposed (through infection or administration) to Neisseriameningitidis or to an antigen composition derived from Neisseriameningitidis in sufficient amounts to elicit protective immunity, or ifexposed, failed to mount a protective immune response. (An example ofthe latter would be an individual exposed at a too young age whenprotective immune responses may not occur. Molages et al., 1994, Infect.Immun. 62: 4419-4424). It is further desirable (but not necessary) thatthe “immunologically naive” individual has also not been exposed to aNeisserial species other than Neisseria meningitidis (or an antigencomposition prepared from a Neisserial species), particularly not to across-reacting strain of Neisserial species (or antigen composition).Individuals that have been exposed (through infection or administration)to a Neisserial species or to an antigen composition derived from thatNeisserial species in sufficient amounts to elicit an immune response tothe epitopes exhibited by that species, are “primed” to immunologicallyrespond to the epitopes exhibited by that species.

B. Preparation of Neisseria meningitidis Fractions and Detection ofAntigens and Antigenic Compositions that Confer Protective Immunity

1. Antigenic Compositions

The various antigenic compositions (e.g. lysed cells, subcellularfractions, MVs and OMVs, or individual antigens and combinations ofantigens detected and isolated as described above and below) that areadministered to an animal (especially a human patient) to induce animmune response are generally obtained by methods known in the art. Insome embodiments, the antigen preparations used to elicit an immuneresponse are prepared by culturing Neisseria meningitidis spp. usingwell-known bacterial culture techniques and preparing a fraction thatcontains antigens that induce protective immunity.

One preferred fraction comprises microvesicles (MV) or blebs that arereleased during culture of said Neisseria meningitidis spp. MVs may beobtained by culturing a strain of Neisseria meningitidis in brothculture medium, separating whole cells from the broth culture medium(e.g. by filtration, or by a low-speed centrifugation that pellets onlythe cells and not the smaller blebs, or the like), and then collectingthe MVs that are present in the cell-free culture medium (e.g. byfiltration, differential precipitation or aggregation of MVs, or by ahigh-speed centrifugation that pellets the blebs, or the like). Strainsfor use in production of MVs can generally be selected on the basis ofthe amount of blebs produced in culture (e.g., bacteria can be culturedin a reasonable number to provide for production of blebs suitable forisolation and administration in the methods described herein). Anexemplary strain that produces high levels of blebs is described in PCTPublication No. WO 01/34642. In addition to bleb production, strains foruse in MV production may also be selected on the basis of NspAproduction, where strains that produce higher levels of NspA may bepreferable (for examples of N. meningitides strains having differentNspA production levels, see, e.g., Moe et al. (1999 Infect. Immun. 67:5664).

A second preferred fraction comprises outer membrane vesicles (OMV)prepared from the outer membrane of a cultured strain of Neisseriameningitidis spp. OMVs may be obtained from a Neisseria meningitidisgrown in broth or solid medium culture, preferably by separating thebacterial cells from the culture medium (e.g. by filtration or by alow-speed centrifugation that pellets the cells, or the like), lysingthe cells (e.g. by addition of detergent, osmotic shock, sonication,cavitation, homogenization, or the like) and separating an outermembrane fraction from cytoplasmic molecules (e.g. by filtration; or bydifferential precipitation or aggregation of outer membranes and/orouter membrane vesicles, or by affinity separation methods using ligandsthat specifically recognize outer membrane molecules; or by a high-speedcentrifugation that pellets outer membranes and/or outer membranevesicles, or the like); outer membrane fractions may be used to produceOMVs.

In the production of MVs or OMVs, it may be preferable to use strainsthat are relatively low producers of endotoxin (lipopolysaccharide, LPS)so as to decrease the need to remove endotoxin from the finalpreparation prior to use in humans. For example, the OMV and/or MV canbe prepared from mutants of these Neisseria strains in whichlipooligosaccharide or other antigens that may be undesirable in avaccine (e.g. Rmp) is reduced or eliminated.

Where desired (e.g., where the strains used to produce MVs or OMVs areassociated with endotoxin or particular high levels of endotoxin), theMVs or OMVs are optionally treated to reduce endotoxin, e.g., to reducetoxicity following administration. Reduction of endotoxin can beaccomplished by extraction with a suitable detergent (for example,BRIJ-96, sodium deoxycholate, sodium lauoylsarcosinate, Empigen BB,Triton X-100, TWEEN 20 (sorbitan monolaurate polyoxyethylene), TWEEN 80,at a concentration of 0.1-10%, preferably 0.5-2%). Where detergentextraction is used, it is preferable to use a detergent other thandeoxycholate. Extraction of OMV and MV preparations with deoxycholateresulted in removal of some non-capsular protein antigens (see FIG. 2).Vaccination of animals with OMV or MV preparations subjected todeoxycholate extraction elicited an immune response that was associatedwith lower titers of bactericidal antibodies compared to vaccinationwith non-deoxycholate-extracted material .

In addition to MVs or OMVs, isolated antigens or particular combinationsof antigens may be used to induce a protective immune response. Theidentity of the isolated antigens or combinations of antigens aredescribed below.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen, as well as any other compatible components,as needed. By “immunologically effective amount” is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group o individual to betreated (e.g., non-human primate, primate, etc.), the capacity of theindividual's immune system to synthesize antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingclinician's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials. Dosage treatmentmay be a single dose schedule or a multiple dose schedule (e.g.,including booster doses). The vaccine may be administered in conjunctionwith other immunoregulatory agents.

The antigen compositions or individual antigens to be administered areprovided in a pharmaceutically acceptable solution such as an aqueoussolution, often a saline solution, or they can be provided in powderform. The compositions may also include an adjuvant. Examples of knownsuitable adjuvants that can be used in humans include, but are notnecessarily limited to, alum, aluminum phosphate, aluminum hydroxide,MF59 (4.3% w/v squalene, 0.5% w/v Tween 80, 0.5% w/v Span 85),CpG-containing nucleic acid (where the cytosine is unmethylated), QS21,MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CT mutants,poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A,interleukins, and the like. For experimental animals, one can useFreund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicantigen.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (W090/14837; Chapter 10 in Vaccine design: the subunitand adjuvant approach, eds. Powell & Newman, Plenum Press 1995),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining MTP-PE) formulated into submicron particles using amicrofluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components such as monophosphorylipid A (MPL),trebalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS DETOX™); (2) saponin adjuvants, such as QS21 or STIMULON™(Cambridge Bioscience, Worcester, Mass.) may be used or particlesgenerated therefrom such as ISCOMs (immunostimulating complexes), whichISCOMS may be devoid of additional detergent e.g. WO00/07621; (3)Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA);(4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL(3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358;(6) combinations of 3dMPL with, for example, QS21 and/oroil-in-wateremulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7)oligonucleotides comprising CpG motif [Krieg Vaccine 2000, 19,618-622;Krieg Curr opin Mol Ther2001 3:15-24; Roman et al., Nat. Med.1997,3,849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis etal, J. Immunol, 1998, 160, 870-876; Chu et al., J. Exp.Med, 1997, 186,1623-1631; Lipford et al, Ear. J. Immunol., 1997, 27, 2340-2344;Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature,1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883;Ballas et al, J. Immunol, 1996, 157, 1840-1845; Cowdery et al, J.Immunol, 1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996, 167,72-78; Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey etal, J. Immunol., 1996, 157,2116-2122; Messina et al, J. Immunol, 1991,147, 1759-1764; Yi et al, J. Immunol, 1996, 157,4918-4925; Yi et al, J.Immunol, 1996, 157, 5394-5402; Yi et al, J. Immunol, 1998, 160,4755-4761; and Yi et al, J. Immunol, 1998, 160, 5898-5906; Internationalpatent applications WO96/02555, WO98/16247, WO98/18810, WO98/40100,WO98/55495, WO98/37919 and WO98/52581] i.e. containing at least one CGdinucleotide, where the cytosine is unmethylated; (8) a polyoxyethyleneether or a polyoxyetbylene ester e.g. WO99/52549; (9) a polyoxyethylenesoibitan ester surfactant in combination with an octoxynol (WO01/21207)or a polyoxyethylene alkyl ether or ester surfactant in combination withat least one additional non-ionic surfactant such as an octoxynol(WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide(e.g. a CpG oligonucleotide) (WO00/62800); (11) an immunostimulant and aparticle of metal salt e.g. WO00/23105; (12) a saponin and anoil-in-water emulsion e.g. WO99/11241; (13) a saponin (e.g.QS21)+3dMPL+IM2 (optionally+a sterol) e.g. WO98/57659; (14) othersubstances that act as immunostimulating agents to enhance the efficacyof the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc.

The antigens may be combined with conventional excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like. Theconcentration of antigen in these formulations can vary widely, and willbe selected primarily based on fluid volumes, viscosities, body weightand the like in accordance with the particular mode of administrationselected and the patient's needs. The resulting compositions may be inthe form of a solution, suspension, tablet, pill, capsule, powder, gel,cream, lotion, ointment, aerosol or the like.

The concentration of immunogenic antigens of the invention in thepharmaceutical formulations can vary widely, i.e. from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

2. Immunization

The MVs, OMVs, isolated antigens, or combinations of antigens of thepresent invention are administered orally, nasally, nasopharyngeally,parenterally, enterically, gastrically, topically, transdermally,subcutaneously, intramuscularly, in tablet, solid, powdered, liquid,aerosol form, locally or systemically, with or without added excipients.Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as Remington's PharmaceuticalScience, 15th ed., Mack Publishing Company, Easton, Pa. (1980).Administration of the MVs, OMVs, isolated antigens, or combinations ofantigens can be performed serially or as a mixture, as described in moredetail below.

It is recognized that the polypeptides and related compounds describedabove, when administered orally, must be protected from digestion. Thisis typically accomplished either by complexing the protein with acomposition to render it resistant to acidic and enzymatic hydrolysis orby packaging the protein in an appropriately resistant carrier such as aliposome. Means of protecting proteins from digestion are well known inthe art.

In order to enhance serum half-life, the antigenic preparations that areinjected may also be encapsulated, introduced into the lumen ofliposomes, prepared as a colloid, or other conventional techniques maybe employed which provide an extended serum half-life of the peptides. Avariety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The preparations may alsobe provided in controlled release or slow-release forms for release andadministration of the antigen preparations as a mixture or in serialfashion.

The compositions are administered to an animal that is at risk fromacquiring a Neisserial disease to prevent or at least partially arrestthe development of disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for therapeutic use will depend on, e.g., the antigencomposition, the manner of administration, the weight and general stateof health of the patient, and the judgment of the prescribing physician.Single or multiple doses of the antigen compositions may be administereddepending on the dosage and frequency required and tolerated by thepatient, and route of administration.

In particular embodiments, the antigen compositions described herein areadministered serially. First, a therapeutically effective dose of afirst antigen composition (e.g. MV, OMV, isolated antigen, orcombinations of antigens, with or without excipients) prepared from afirst Neisserial strain is administered to an individual. The firstantigenic composition is generally administered in an amount effectiveto elicit a immune response (e.g., activation of B and/or T cells).Amounts for the initial immunization generally range from about 0.001 mgto about 1.0 mg per 70 kilogram patient, more commonly from about 0.001mg to about 0.2 mg per 70 kilogram patient. Dosages from 0.001 up toabout 10 mg per patient per day may be used, particularly when theantigen is administered to a secluded site and not into the bloodstream, such as into a body cavity or into a lumen of an organ.Substantially higher dosages (e.g. 10 to 100 mg or more) are possible inoral, nasal, or topical administration.

After administration of the first antigen composition, a therapeuticallyeffective dose of a second antigen composition (e.g. MV, OMV, isolatedantigen, or combinations of antigens, with or without excipients)prepared from a second Neisserial strain is administered to anindividual after the individual has been immunologically primed byexposure to the first antigen composition. The booster may beadministered days, weeks or months after the initial immunization,depending upon the patient's response and condition. The existence of animmune response to the first antigen composition may be determined byknown methods (e.g. by obtaining serum from the individual before andafter the initial immunization, and demonstrating a change in theindividual's immune status, for example an immunoprecipitation assay, oran ELISA, or a bactericidal assay, or a Western blot, or flow cytometricassay, or the like) and/or demonstrating that the magnitude of theimmune response to the second injection is higher than that of controlanimals immunized for the first time with the composition of matter usedfor the second injection (e.g. immunological priming). Immunologicpriming and/or the existence of an immune response to the first antigencomposition may also be assumed by waiting for a period of time afterthe first immunization that, based on previous experience, is asufficient time for an immune response and/or priming to have takenplace—e.g. 2, 4, 6, 10 or 14 weeks. Boosting dosages of the secondantigen composition are typically from about 0.001 mg to about 1.0 mg ofantigen, depending on the nature of the immunogen and route ofimmunization.

In certain preferred embodiments, a therapeutically effective dose of athird antigen composition prepared from a third Neisserial strain isadministered to an individual after the individual has been primedand/or mounted an immune response to the second antigen composition. Thethird booster may be administered days, weeks or months after the secondimmunization, depending upon the patient's response and condition. Theexistence of priming and/or an immune response to the second antigencomposition may be determined by the same methods used to detect animmune response to the second antigen composition. The existence ofpriming and/or an immune response to the second antigen composition mayalso be assumed by waiting for a period of time after the secondimmunization that, based on previous experience, is a sufficient timefor an immune response to have taken place—e.g. 2, 4, 6, 10 or 14 weeks.Boosting dosages of the second antigen composition are typically fromabout 0.001 mg to about 1.0 mg of antigen, depending on the nature ofthe immunogen and route of immunization. The present invention furthercontemplates the use of a fourth, fifth, sixth or greater boosterimmunization, using either a fourth, fifth or sixth strain of Neisseriameningitidis or any of the first, second, or third strains, or otherstrain that is genetically diverse with respect to at least one of thefirst, second, and third strains.

Where administration of antigenic compositions prepared from the first,second, and (optionally, but preferably) third strains is serial, theorder of administration of the compositions can be varied. For example,the order of administration of OMV and/or MV within these serialadministration steps can be varied. For example, the following orders ofadministration are within the scope of the invention (from left toright, with the third administration being optional): OMV-OMV-OMV;OMV-OMV-MV; OMV-MV-MV; MV-MV-MV; MV-MV-OMV; MV-OMV-OMV; OMV-MV-OMV; andMV-OMV-MV. Preferably, the order of administration is MV-MV-OMV.

In other embodiments the first, second, and (optionally) third antigencompositions are administered as a mixture. In related embodiments, thefirst and second antigen compositions are administered as a mixture, andthe third antigen composition is administered subsequently.

The mixtures is administered in an amount effective to elicit an immuneresponse, particularly a humoral immune response, in the host. Amountsfor the immunization of the mixture generally range from about 0.001 mgto about 1.0 mg per 70 kilogram patient, more commonly from about 0.001mg to about 0.2 mg per 70 kilogram patient. Dosages from 0.001 up toabout 10 mg per patient per day may be used, particularly when theantigen is administered to a secluded site and not into the bloodstream, such as into a body cavity or into a lumen of an organ.Substantially higher dosages (e.g. 10 to 100 mg or more) are possible inoral, nasal, or topical administration. The initial administration ofthe mixture can be followed by booster immunization of the same ofdifferent mixture, with at least one booster, more usually two boosters,being preferred.

In certain preferred embodiments, the first and second Neisserialstrains are genetically diverse to one another, e.g., the strains belongto different PorB serotypes and/or PorA serosubtypes; and may alsooptionally belong to different capsular serogroups. Furthermore, thesecond and third Neisserial strains are genetically diverse to oneantoher, e.g., the strains belong to different serotypes and/orserosubtypes; may also optionally belong to different serogroups. Thethird Neisserial strain is preferably genetically diverse with respectto the first and second strains, but may, in some embodiments, not begenetically diverse with respect to the first strain. For example, theserotype and/or serosubtype of the third Neisserial strain shouldpreferably be different from the first and second strain but it may bethe same as the first strain.

The present invention also specifically contemplates that antigencompositions from other members of the genus Neisseria may beadministered as described herein to generate protective immunity againstNeisseria meningitidis. For example, Neisseria lactamica, anonpathogenic non-encapsulated commensal member of the genus Neisseriathat is commonly found in the human nasopharynx, encompasses strainswhich have many antigens present on N. meningitidis and, therefore, alsomay be used to prepare one of the immunogens envisioned in thisinvention. Thus, MVs and OMVs from the nonpathogenic Neisseria lactamicamay be used to prime or elicit a protective immune response againstNeisseria meningitidis (or against other pathogenic Neisseria such asNeisseria gonorrhea). This may be accomplished by initiallyadministering an antigen composition (e.g., MV or OMV) from Neisserialactamica, followed by administering a second and optionally a thirdantigen composition from Neisseria meningitidis (or Neisseriagonorrhea). The invention specifically contemplates also that antigencompositions from Neisseria lactamica strains be used for the initial,second and any subsequent administrations, wherein each lactamica strainhas a different serotype and/or serosubtype as the others.

The invention also contemplates that the antigen compositions used atany step in the immunization protocol may be obtained from one or morestrains of bacteria (especially Neisseria lactamica or Neisseriameningitidis) that are genetically engineered by known methods (see,e.g. U.S. Pat. No. 6,013,267) to express one or more nucleic acids thatencode one or more molecules of interest, particularly molecules thatelicit or enhance a protective immune response. The nucleic acids may,for example, encode Porin A, Porin B, NspA, pilin, or other Neisserialproteins. Other exemplary nucleic acids include those that encodeNeisserial proteins immunoprecipitated with anti-sera produced followingvaccination with the CHORI vaccine, particularly those proteins havingapparent molecular masses of about 80 kDa, about 59.5 kDa, about 40.7kDa, about 39.6 kDa, about 33 kDa, about 27.9 kDa, and 14.5 kDa, orantigenic fragments thereof. Further exemplary nucleic acids includethose that encode Neisserial proteins detected by Western blot withanti-sera produced following vaccination with the CHORI vaccine,particularly those proteins having apparent molecular masses of about 53kDa-57 kDa; about 46-47 kDa, about 33 kDa, about 20 kDa to 21 kDa; andabout 18 kDa. The nucleic acids may encode any of the above proteinsthat is truncated, or altered to include or delete a glycosylation site,or to include or delete any epitope, or to increase the expression ofany of the above proteins. Of particular interest are antigenicfragments of such proteins. In addition, the antigen compositions of theinvention can comprise additional antigens of N. meningiditis such asthose exemplified in PCT Publication Nos. WO 99/24578, WO 99/36544; WO99/57280, WO 00/22430, and WO 00/66791, as well as antigenic fragmentsof such proteins.

An important aspect of the present invention is that the antigencompositions used to prime and boost a broad protective immunity againstNeisseria meningitidis are prepared from strains of Neisseria thatpossess variant immunodominant antigens (the main antigens that areroutinely detected by antisera from different host animals that havebeen infected with Neisseria; representative examples include Porin A,Porin B, pilin, NspA etc.). In the examples described in the Examplessection below, the strains vary with respect to either PorA or PorB, asevinced by their serotype or serosubtype.

The strains also may vary with respect to the capsule molecule, asreflected by their serogroup.

Serotype and serosubtype classification is currently determined bydetecting which of a panel of known monoclonals, which are known torecognize specific Porin molecules, bind to an unknown strain (Sacchi etal., 1998, Clin. Diag. Lab. Immunol. 5:348, see Tables 8 and 9 forpartial lists of monoclonals). It is probable that other suchmonoclonals will be identified. The use of any novel serotypes andserosubtypes which may be defined by any new monoclonals arespecifically contemplated by the invention. In addition, serotypes andserosubtypes may be defined, not only by interaction with monoclonalantibodies, but also structurally by the absence and/or presence ofdefined peptide residues and peptide epitopes (Sacchi et al., 2000, J.Infect. Dis. 182:1169). Serotype and serosubtype classification schemesthat are based on structural features of the Porins (known or that maybe discovered at a later date) are specifically encompassed by theinvention.

One purpose and effect of serial administration of antigen compositionsfrom different strains is to potentiate an immune response to antigensand epitopes that are typically not immunodominant, particularlynon-immunodominant epitopes that exhibit less genetic variability thanthe known immunodominant epitopes. The invention specificallyencompasses the serial administration of antigen compositions fromNeisserial strains that differ with respect to immunodominant antigensother than the Porins (e.g., phospholipids, polysaccharides,lipopolysaccharides, pilins, OmpA, Opa, Opc, etc.).

The antigen compositions are typically administered to a mammal that isimmunologically naive with respect to Neisseria meningitidis. In aparticular embodiment, the mammal is a human child about five years oryounger, and preferably about two years old or younger, and the antigencompositions are administered at any one or more of the following times:two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or oneyear or 15, 18, or 21 months after birth, or at 2, 3, 4, or 5 years ofage.

In general, administration to any mammal is preferably initiated priorto the first sign of disease symptoms, or at the first sign of possibleor actual exposure to Neisseria.

Where particular immunogenic peptides that give rise to protectiveimmunity are identified as described above and below, these antigens maybe directly administered instead of MVs or OMVs. Where the identifiedantigens are peptides, the DNA encoding one or more of the peptides ofthe invention can also be administered to the patient. This approach isdescribed, for instance, in Wolff et al., Science 247: 1465-1468 (1990),as well as U.S. Pat. Nos. 5,580,859 and 5,589,466.

3. Detection of Immunogenic Antigens

Subcellular fractions such as MVs and OMVs contain many antigens thatmay give rise to an immune response (see, e.g., FIG. 2 which depicts anelectrophoretic gel of several such fractions). However, not everyantigen in a preparation may elicit either a humoral response orprotective immunity against a disease caused by a Neisseria meningitidisspp. Thus, the present invention also relates to individual antigensand/or combinations of antigens that induce protective immunity. Anotherobjective is to use the identified antigens to formulate antigencompositions that may be used to elicit protective immunity against aNeisserial disease.

Antisera or mAbs are obtained or produced from mammals that are inducedby the methods of the present invention to exhibit protective immunityagainst a disease caused by a Neisseria meningitidis spp. The antiseraor mAbs are used to detect their corresponding Neisserial antigens, andthe these antigens identified and isolated based on theirphysicochemical properties (class of molecule: peptide, nucleic acid,etc.; molecular mass, charge, chemical composition, etc.) or amino acidsequence. The isolated antigens (or immunologically effective portionsthereof) can then be administered to mammals, singly and/or incombination, as described above, or as recombinant proteins (orimmunologically effective fragments thereof) to assess the extent towhich they induce protective immunity. The present inventionspecifically contemplates serially administering antigens isolated fromone strain or different strains of Neisseria, or administering suchantigens in a mixture comprising one or more, usually two or more, moreusually three or more, still more usually four to six or more antigens.

Exemplary Neisserial antigens suitable for administration as describedmay include, but are not necessarily limited to those proteinsimmunoprecipitated with anti-sera produced following vaccination withthe CHORI vaccine, particularly those proteins having apparent molecularmasses of about 80 kDa, about 59.5 kDa, about 40.7 kDa, about 39.6 kDa,about 33 kDa, about 27.9 kDa, and 14.5 kDa, or antigenic fragmentsthereof. The antigens administered may include at least one of theseantigens, or may include a combination of these antigens (e.g., at leasttwo, at least three, at least four, or more).

Further exemplary Neisserial antigens suitable for administration asdescribed may include, but are not necessarily limited to those proteinsdetected by Western blot with anti-sera produced following vaccinationwith the CHORI vaccine, particularly those proteins having apparentmolecular masses of about 53 kDa-57 kDa; about 46-47 kDa, about 33 kDa,about 20 kDa to 21 kDa; and about 18 kDa, or antigenic fragmentsthereof. The antigens administered may include at least one of theseantigens, or may include a combination of these antigens (e.g., at leasttwo, at least three, at least four, or more).

It should be noted that references made to molecular masses of proteinsrefer to apparent molecular mass as determined using SDS-PAGE under theconditions described. It will be readily apparent to the ordinarilyskilled artisan upon reading the present specification that theseapparent molecular masses may vary for the same protein between twodifferent experiments (e.g., using different gels or differentpreparations, when isolated from two different strains (e.g., due topolymorphisms between strains due to amino acid substitutions,deletions, and/or insertions, post-translational modifications, and thelike)), and further that different proteins may appear to have the sameapparent molecular mass.

Antigens that elicit protective immunity are detected by known methods:for example, immunoassay, immunoprecipitation, affinity chromatography,Western blots, etc. Examples of such methods are described below.

(a) Detection of Antigens by Immunoassay

A variety of immunoassay formats are used to characterize antisera andantibodies specifically immunoreactive with a particular antigen orantigenic composition, and also to measure the strength of an immuneresponse to a particular antigen or antigenic composition. The firststep is generally producing antiserum or an antibody preparation thatbinds to the antigen or antigenic composition.

(1) Production of Immune Antisera and Specific Polyclonal or MonoclonalAntibodies

Methods of producing polyclonal and monoclonal antibodies used in theseassays are known to those of skill in the art. See, e.g., Coligan(1991), CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y.; and Harlowand Lane (1989), ANTIBODIES: A LABORATORY MANUAL, Cold Spring HarborPress, N.Y.; Stites et al. (eds.) 1997 MEDICAL IMMUNOLOGY 9th ed.McGraw-Hill Professional Publishing, New York, N.Y., and referencescited therein; Goding (1986), MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975), Nature, 256: 495-497. For example, in order to produceantisera for use in an immunoassay, a composition that contains antigensfrom Neisseria meningitidis spp. is given alone or mixed with anadjuvant and injected into an animal of choice (e.g. a mouse, rat,rabbit, pig, goat, cow, horse, chicken, etc.) according to any of theprotocols described herein.

The animal's immune response to the immunogen preparation is monitoredby taking test bleeds and determining the titer of reactivity ofserially diluted aliquots of serum to serially diluted aliquots of theantigenic composition. Polyclonal antisera with a titer of 10⁴ orgreater are selected and tested for their cross reactivity againstnon-immunogenic controls, using a competitive binding immunoassay.Specific monoclonal and polyclonal antibodies and antisera will usuallybind with a dissociation constant (K_(D)) of at least about 0.1 mM, moreusually at least about 1 μM, preferably at least about 0.1 μM or better,and most preferably, 0.1 μM or better.

(2) Monoclonal Antibodies

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. Supra, and references cited therein;Harlow and Lane, Supra; Goding Supra; and Kohler and Milstein. Supra.Summarized briefly, this method proceeds by injecting an animal with animmunogenic preparation. The animal is then sacrificed and cells takenfrom its spleen, which are fused with myeloma cells. The result is ahybrid cell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secrete a single antibody species to the immunogen. Inthis manner, the individual antibody species obtained are the productsof immortalized and cloned single B cells from the immune animalgenerated in response to a specific site recognized on the immunogenicsubstance.

Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe antigen, and yield of the monoclonal antibodies produced by suchcells is enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate (preferably mammalian) host.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse et al. (1989)Science 246: 1275-1281; Ward et al. (1989) Nature 341: 544-546; andVaughan et al. (1996) Nature Biotechnology 14: 309-314).

(3) Immunoassays

Once an immune serum or a monoclonal antibody that recognizes one ormore Neisserial antigens is obtained, it may be used to performimmunoassays. For a review of immunological and immunoassay proceduresin general, see D. Stites and A. Terr, (eds.), Supra. Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in ENZYME IMMUNOASSAY, E.T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980); “Practice and Theoryof Enzyme Immunoassays,” P. Tijssen, in LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY, Elsevier Science Publishers B. V.Amsterdam (1985); and Harlow and Lane, supra, each of which isincorporated herein by reference.

For example, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with an antigen. SeeHarlow and Lane, Supra.

Immunoabsorbed and/or pooled antisera (or monoclonal antibodies) arealso used in a direct or competitive binding immunoassay. The lattercompares the binding of a second antigen composition (e.g. MVs, OMVs,isolated antigens or antigen compositions from an unknown or a knowndifferent Neisserial strain) to that of the reference antigencomposition used to elicit protective immunity. In order to make thiscomparison in the competitive assay, the two antigen preparations areeach assayed at a wide range of concentrations and the amount of eachmolecule required to inhibit 50% of the binding of the antisera to theimmobilized reference antigen preparation is determined. If the amountof the second protein required is less than 10 times the amount of thereference peptide used to make the antibody, then the second protein issaid to specifically bind to an antibody generated to the referenceantigen preparation.

(b) Western Blots

Western blot analysis generally comprises separating sample products bygel electrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support (such as a nitrocellulosefilter, a nylon filter, or derivatized nylon filter), and incubating thesample with labeling antibodies that specifically bind to the analyteprotein. The labeling antibodies specifically bind to analyte on thesolid support. These antibodies are directly labeled, or alternativelyare subsequently detected using labeling agents such as antibodies (e.g.labeled sheep anti-mouse antibodies where the antibody to an analyte isa murine antibody) that specifically bind to the labeling antibody.

4. Purification of Immunogenic Antigens

The antigens can be isolated (separated from one or more molecules withwhich the antigen is associated in vivo) and purified (a purifiedantigen, e.g. a protein, preferably exhibits essentially a single bandon an electrophoretic gel for each dissociable subunit of the antigen)and used to elicit protective immunity.

Individual antigens, especially proteins and peptide fragments thereof,can be purified by any of a variety of known techniques, including, forexample, reverse phase high-performance liquid chromatography (HPLC),ion-exchange or immunoaffinity chromatography, separation by size, orelectrophoresis (see, generally, Scopes, R. Protein Purification,Springer-Verlag, N.Y. (1982)). For example, antigens from Neisseriameningitidis spp. that are recognized by broad spectrum antiseraobtained after serial injections of OMVs and/or MVs obtained fromdifferent Neisseria meningitidis spp. are obtained by using broadspectrum antisera to generate enriched antigen preparations. Isolatedantigens may also be prepared by immunoprecipitating a fraction obtainedfrom Neisseria meningitidis spp. The antigens may also be isolated byconjugating immune antisera or monoclonal antibodies to a column andperforming affinity chromatography. The source of the antigens may be awhole cell lysate obtained by known methods, for example by sonication,or alternatively by exposure to an ionic or nonionic detergent, or thesource may be MVs or OMVs from a Neisserial strain.

5. Peptide Antigens

Furthermore, once the identity of protein antigens and/or specificpeptide epitopes is established, antigen preparations from Neisseriameningitidis spp. suitable for inducing protective immunity in thepresent invention can be generated by synthesizing peptides byconventional techniques, and injecting synthetic peptide preparationsinto a mammal. Techniques for peptide synthesis are well known in theart. See, e.g., Stewart and Young, Solid Phase Peptide Synthesis(Rockford, Ill., Pierce), 2d Ed. (1984) and Kent, 1988, Annu. Rev.Biochem. 57:957.

Alternatively, nucleic acid sequences which encode the particularpolypeptide may be cloned and expressed to provide the peptide. Standardtechniques can be used to obtain and screen nucleic acid libraries toidentify sequences encoding the desired sequences (see Sambrook et al.,Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989), or nucleic acids that encode desiredpeptides may be synthesized by known methods. Fusion proteins (thoseconsisting of all or part of the amino acid sequences of two or moreproteins) can be recombinantly produced. In addition, using in vitromutagenesis techniques, unrelated proteins can be mutated to comprisethe appropriate sequences.

It will be understood that the immunogenic antigens of the presentinvention may be modified to provide a variety of desired attributes,e.g. improved pharmacological characteristics, while increasing or atleast retaining substantially all of the biological activity of theunmodified peptide. For instance, the peptides can be modified byextending, decreasing the amino acid sequence of the peptide.Substitutions with different amino acids or amino acid mimetics can alsobe made.

The peptides employed in the subject invention need not be identical tothose disclosed in the Examples section below (e.g., with respect tomolecular weight), so long as the subject peptides are able to induce animmune response against the desired antigen molecule. Thus, one of skillwill recognize that a number of conservative substitutions (described inmore detail below) can be made without substantially affecting theactivity of the peptide.

Single amino acid substitutions, deletions, or insertions can be used todetermine which residues are relatively insensitive to modification.Substitutions are preferably made with small, relatively neutralmoieties such as Ala, Gly, Pro, or similar residues. The effect ofsingle amino acid substitutions may also be probed using D-amino acids.The numbers and types of residues which are substituted or added dependon the spacing necessary between essential contact points and certainfunctional attributes which are sought (e.g. hydrophobicity versushydrophilicity). Increased immunogenicity may also be achieved by suchsubstitutions, compared to the parent peptide. In any event, suchsubstitutions should employ amino acid residues or other molecularfragments chosen to avoid, for example, steric and charge interferencewhich might disrupt binding.

The substituting amino acids, however, need not be limited to thosenaturally occurring in proteins, such as L-α-amino acids, or theirD-isomers. The peptides may be substituted with a variety of moietiessuch as amino acid mimetics well known to those of skill in the art.(See, e.g., U.S. Pat. No. 6,030,619).

The individual residues of the immunogenic antigenic polypeptides can beincorporated in the peptide by a peptide bond or peptide bond mimetic. Apeptide bond mimetic of the invention includes peptide backbonemodifications well known to those skilled in the art. Such modificationsinclude modifications of the amide nitrogen, the α-carbon, amidecarbonyl, complete replacement of the amide bond, extensions, deletionsor backbone crosslinks. See, generally, Spatola, Chemistry andBiochemistry of, Amino Acids, Peptides and Proteins, Vol. VII (Weinsteined., 1983).

Several peptide backbone modifications are known. These include ψ[CH₂S],Ψ[CH₂NH], Ψ[CSNH₂], Ψ[NHCO], Ψ[COCH₂] and Ψ[(E) or (Z) CH CH]. Thenomenclature used above follows that suggested by Spatola, above. Inthis context, Ψ indicates the absence of an amide bond. The structurethat replaces the amide group is specified within the brackets.

Amino acid mimetics may also be incorporated in the peptides. An “aminoacid mimetic” as used here is a moiety other than a naturally occurringamino acid that conformationally and functionally serves as a substitutefor an amino acid in a polypeptide of the present invention. Such amoiety serves as a substitute for an amino acid residue if it does notinterfere with the ability of the peptide to illicit an immune responseagainst the appropriate antigen. Amino acid mimetics may includenon-protein amino acids, such as β-γ-δ-amino acids, β-γ-δ-imino acids(such as piperidine-4-carboxylic acid), as well as many derivatives ofL-α-amino acids. A number of suitable amino acid mimetics are known tothe skilled artisan; they include cyclohexylalanine,3-cyclohexylpropionic acid, L-adamantyl alanine, adamantylacetic acidand the like. Peptide mimetics suitable for peptides of the presentinvention are discussed by Morgan and Gainor, (1989) Ann. Repts. Med.Chem. 24: 243-2526.

As noted above, the peptides employed in the subject invention need notbe identical, but may be substantially identical, to the correspondingsequence of the target antigen. Therefore, the peptides may be subjectto various changes, such as insertions, deletions, and substitutions,either conservative or non-conservative, where such changes mightprovide for certain advantages in their use. The polypeptides of theinvention can be modified in a number of ways so long as they comprise asequence substantially identical (as defined below) to a sequence in thetarget region of the antigen.

Alignment and comparison of relatively short amino acid sequences (lessthan about 30 residues) is typically straightforward. Comparison oflonger sequences may require more sophisticated methods to achieveoptimal alignment of two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. (USA) 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e. resulting in the highest percentage of sequencesimilarity over the comparison window) generated by the various methodsis selected.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In a mostpreferred embodiment, the sequences are substantially identical over theentire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al, supra).

These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Polypeptides encompassed by the invention typically comprise at leastabout 10 residues and more preferably at least about 15 residues,preferably from a domain of the antigen that is exposed to the immunesystem. In certain embodiments the peptides will not exceed about 50residues and typically will not exceed about 30 residues.

The immunogenic peptides are conformationally constrained. Means forachieving this are well known in the art (see, e.g., Hruby and Bonner inMethods in Molecular Biology, Volume 35: Peptide Synthesis Protocols,Pennington and Dunn eds (Humana Press, Totowa, N.J., 1994). A preferredmeans for preparing conformationally constrained peptides is throughcyclization. Any method commonly used to produce cyclized oligopeptidescan be used to produce the peptides of the invention. For example, incertain embodiments the peptides will include cysteine residues at bothtermini, which allow the production of cyclic peptides through disulfidelinkages. Treatment of such a peptide with an oxidizing agent such asoxygen, iodine or similar agent will produce a cyclic peptide which maybe further purified using chromatographic or other methods of chemicalpurification. Construction of cyclic peptides can also be accomplishedthrough thioether linkages. For instance, N-bromoacetyl-derivatizedpeptides can be reacted with sulfhydryl-containing residues, such ascysteine. Cyclization occurs by reaction of the free sulfhydryl ofcysteine in the peptide with the bromoacetyl group to form a thioetherlinkage (Robey et al., Anal. Biochem. 177: 373-7 (1989) and U.S. Pat.No. 5,066,716).

Other methods of constructing cyclic peptides are known to those skilledin the art. These include side chain-side chain, side chain-main chainand main chain-main chain cyclizations. In addition, linkers can be usedto join the amino and carboxyl termini of a peptide. The linker iscapable of forming covalent bonds to both the amino and carboxylterminus. Suitable linkers are well known to those of skill in the artand include, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. The linkersmay be joined to the carboxyl and amino terminal amino acids throughtheir side groups (e.g. through a disulfide linkage to cysteine) orthrough the alpha carbon amino and carboxyl groups of the terminal aminoacids.

For a general discussion of suitable methods for cyclization, see Hrubyand Bonner in Methods in Molecular Biology, Volume 35: Peptide SynthesisProtocols, Pennington and Dunn eds (Humana Press, Totowa, N.J., 1994).For instance, cyclizations may include formation of carba analogs andthioethers (Lebl et al. in Peptides 1986 Proceedings of the 19thEuropean Peptide Symposium pp. 341-344; Robey et al., Anal. Biochem.177: 373-7 (1989) and U.S. Pat. No. 5,066,716), bis-thioethers (Mosberget al. JACS 107: 2986-2987 (1985)), azopeptides (Siemion et al. Mol.Cell. Biochem. 34: (1991)), and other cyclic structures, such asbridging structures (Charpentier, M., et al., J. Med. Chem. 32(6):1184-1190 (1989), Thaisrivongs, S., et al., J. Med. Chem. 34(4): 127(1991) and Ozeki, E., et al., Int. J. Peptide Protein Res. 34:111(1989)). Cyclization from backbone-to-backbone positions may also beused.

Bridging is a special type of cyclization in which distant sites in apeptide are brought together using separate bridging molecules orfragments. Bridging molecules may include, for example, succinicanhydride molecules (Charpentier, B., et al., supra), andcarboxymethylene fragments (Thaisrivongs, S., et al., supra). Bridgingby metals can also be used (Ozeki, E., et al., supra).

In some embodiments, the peptides include two or more cystine residues.The cystines can be substituted or added within the peptide or at eitherterminus. The position of the cystines is not critical so long asdisulfide linkages can form between them which allow the production ofcyclic peptides. For example, treatment of such a peptide with anoxidizing agent such as oxygen, iodine or similar agent will produce acyclic peptide which may be further purified using chromatographic orother methods of chemical purification.

Additional embodiments include peptides containing antigenic sequencesof protein sequences that have been incorporated into independentlyfolding peptides (Regan & DeGrado, 1988, Science 241:976; Mutter, 1988,TIBS 13:260; Kamtekar et al., 1993, Science 262:1680; Sieber & Moe,1996, Biochemistry 35:181; Butcher & Moe, 1996, Proc. Natl. Acad. Sci.USA 93:1135; FitzGerald et al., 1998, Biochemistry 273:9951). Theindependently folding peptides may be naturally occurring or of de novodesign.

Peptides capable of eliciting protective immunity similar to that of theCHORI vaccine might also be obtained by using monoclonal antibodiesproduced by immunization with CHORI antigen or the like to selectmolecular mimetics from phage display peptide libraries or othercombinatorial libraries such as small molecules or nucleic acids.

In addition to use of peptides, antibodies raised against peptides ofthe invention can be used to inhibit inflammatory responses. Antibodiescan be raised to the peptides of the present invention using techniqueswell known to those of skill in the art. Anti-idiotypic antibodies canalso be generated. The following discussion is presented as a generaloverview of the techniques available; however, one of skill willrecognize that many variations upon the following methods are known.

Frequently, the peptides and antibodies of the invention will be labeledby joining, either covalently or non-covalently, a substance whichprovides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced. SeeCabilly, U.S. Pat. No.4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

6. Passive Immunity

Immunoprotective antibodies that recognize Neisserial epitopes can alsobe administered to an organism (e.g. a human patient) to induce passiveimmunity against a Neisserial disease, either to prevent infection ordisease from occurring, or as a therapy to improve the clinical outcomein patients with established disease (e.g. decreased complication ratesuch as shock, decreased mortality rate, or decreased morbidity, such asdeafness).

Antibodies administered to an organism other than the species in whichthey are raised are often immunogenic. Thus, for example, murine orporcine antibodies administered to a human often induce an immunologicresponse against the antibody. The immunogenic properties of theantibody are reduced by altering portions, or all, of the antibody intocharacteristically human sequences thereby producing chimeric or humanantibodies, respectively.

Chimeric antibodies are immunoglobulin molecules comprising a human andnon-human portion. More specifically, the antigen combining region (orvariable region) of a humanized chimeric antibody is derived from anon-human source (e.g. murine), and the constant region of the chimericantibody (which confers biological effector function to theimmunoglobulin) is derived from a human source. The chimeric antibodyshould have the antigen binding specificity of the non-human antibodymolecule and the effector function conferred by the human antibodymolecule. A large number of methods of generating chimeric antibodiesare well known to those of skill in the art (see, e.g., U.S. Pat. Nos.5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847,5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235,5,075,431 and 4,975,369). An alternative approach is the generation ofhumanized antibodies by linking the CDR regions of non-human antibodiesto human constant regions by recombinant DNA techniques. See Queen etal., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989) and WO 90/07861.

In one preferred embodiment, recombinant DNA vector is used to transfecta cell line that produces an antibody against a peptide of theinvention. The novel recombinant DNA vector contains a “replacementgene” to replace all or a portion of the gene encoding theimmunoglobulin constant region in the cell line (e.g. a replacement genemay encode all or a portion of a constant region of a humanimmunoglobulin, or a specific immunoglobulin class), and a “targetsequence” which allows for targeted homologous recombination withimmunoglobulin sequences within the antibody producing cell.

In another embodiment, a recombinant DNA vector is used to transfect acell line that produces an antibody having a desired effector function(e.g. a constant region of a human immunoglobulin), in which case, thereplacement gene contained in the recombinant vector may encode all or aportion of a region of an antibody and the target sequence contained inthe recombinant vector allows for homologous recombination and targetedgene modification within the antibody producing cell. In eitherembodiment, when only a portion of the variable or constant region isreplaced, the resulting chimeric antibody may define the same antigenand/or have the same effector function yet be altered or improved sothat the chimeric antibody may demonstrate a greater antigenspecificity, greater affinity binding constant, increased effectorfunction, or increased secretion and production by the transfectedantibody producing cell line, etc.

In another embodiment, this invention provides for fully humanantibodies. Human antibodies consist entirely of characteristicallyhuman polypeptide sequences. The human antibodies of this invention canbe produced by a wide variety of methods (see, e.g., Larrick et al.,U.S. Pat. No. 5,001,065). In one embodiment, the human antibodies of thepresent invention are produced initially in trioma cells (descended fromthree cells, two human and one mouse). Genes encoding the antibodies arethen cloned and expressed in other cells, particularly non-humanmammalian cells. The general approach for producing human antibodies bytrioma technology has been described by Ostberg et al. (1983), Hybridoma2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S.Pat. No. 4,634,666. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

Methods for producing and formulation antibodies suitable foradministration to a subject (e.g., a human subject) are well known inthe art. For example, antibodies can be provided in a pharmaceuticalcomposition comprising an effective amount of an antibody and apharmaceutical excipients (e.g., saline). The pharmaceutical compositionmay optionally include other additives (e.g., buffers, stabilizers,preservatives, and the like). An effective amount of antibody isgenerally an amount effective to provide for protection againstNeisserial disease or symptoms for a desired period, e.g., a period ofat least about 2 days to 10 days or 1 month to 2 months).

7. Diagnostic Assays

The antigens or antibodies of the invention can also be used fordiagnostic purposes. For instance, peptides can be used to screenpre-immune and immune sera to ensure that the vaccination has beeneffective. Antibodies can also be used in immunoassays to detect thepresence of particular antigen molecules associated with Neisserialdisease.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

A. Membrane Preparations

Strains for preparation of OMVS and MVs were selected on the basis ofsergroup, serotype, and serosubtype. Strains to be used in thepreparation of MVs were selected for relatively high levels of blebbingand expression of NspA (see Moe et al. (1999 Infect. Immun. 67: 5664).Exemplary strains used in preparation of OMVs and MVs have beendeposited with the American Type Culture Collection (ATCC; see below).Strains that produce high levels of blebs, which strains areparticularly useful in the MV production, can be selected or are knownin the art (see, e.g., WO 01/34642).

The meningococcal strain frozen at −80° C. in aqueous −2% skim milk(w/v) was subcultured on a commercial chocolate agar plate (Remel,Laztakas, Kans.). After overnight growth at 37° C. in 4% CO₂, severalcolonies were selected to inoculate ˜7 ml of sterile Mueller-Hintonbroth to an OD_(620 nm) of 0.1. The culture was incubated at 37° C., 4%CO₂ with rocking until the OD_(620 nm) reaches 0.6-0.8 (two to threehours). Two to three 7 ml starter cultures were then used to inoculate500 ml of Mueller-Hinton broth. The larger culture was grown to anOD_(620 nm) of 0.9-1.0 at 37° C. with vigorous shaking. Phenol is addedto the culture to a final concentration of 0.5% (w/v) and the mixture isleft at 4° C. overnight to inactivate the bacteria. The cells were thenpelleted by centrifugation (11,000×g) for 30 min. at 4° C. The cellpellets were frozen at −20° C. until used for preparation of outermembrane protein vesicles (OMV).

Microvesicles (MV) were harvested from the phenol-treated cell culturesupernatant by adding solid ammonium sulfate (390 g/l finalconcentration) slowly with stirring. After the ammonium sulfate wasadded and completely dissolved, the mixture was left at 4° C. overnight.The precipitated MVs were then collected by centrifugation at 11,000×gfor 30 min. The precipitated MV pellet was resuspended in 0.04 volume ofPBS and centrifuged again at 16,000×g for 15 min. at 4° C. The pelletwas discarded and the MVs, which remain in the supernatant, werecollected by centrifugation at 100,000×g for 2 hrs. at 4° C. The finalpellet was resuspended in 0.01 volume (i.e. 5 ml per 500 ml of culture)of water (“MV” vaccine preparation). Alternatively, the pellet wasresuspended in 0.1M Tris.HCl, pH 8.6, containing 10 mM EDTA and 0.5%(w/v) sodium deoxycholate (˜3 ml/500 ml cell culture). After stirring(30 min), the mixture was centrifuged (125,000×g, 2 hrs, 4° C.). Thesupernatant was discarded and the pellet resuspended in 1 ml of 3%sucrose (“DOC MV” vaccine preparation). The protein concentration of theMV and DOC MV preparations was determined by BCA assay (Pierce ChemicalCo., Rockford, Ill.). The MV and DOC MV suspensions were then frozen ondry ice and stored at −20° C. until used for immunization.

Outer membrane vesicles (OMV) were prepared by the method of Zollingeret al. (1979 J. Clin. Invest. 63: 836-848). The frozen cell pellet wasresuspended in 10 ml of 0.05 M Tris.HCl buffer, pH 7.4 containing 0.15 MNaCl and 0.01 M EDTA then heated to 56° C. for 30 min. followed bycooling on ice. The cell suspension was then sonicated on ice withseveral 15-second bursts using a microprobe sonifier (Branson, Danbury,Conn.). Cell debris was removed by centrifugation at 16,000×g for 15min., and the outer membrane vesicles (OMVs) in the supernatant wereobtained by ultracentrifugation at 100,000×g for 2 hrs. at 4° C. The OMVpellet was resuspended in 2 ml of water (“OMV” vaccine preparation).Alternatively, the frozen cell pellet was resuspended in 0.1M Tris.HCl,pH 8.6, containing 10 mM EDTA and 0.5% (w/v) sodium deoxycholate. Afterstirring for 30 min. at ambient temperature, the mixture was centrifuged(20,000×g, 30 min., 4° C.). The supernatant was retained and the pelletwas reextracted and centrifuged again with one third volume of the samebuffer. The supernatants from both extractions were combined andcentrifuged (125,000×g, 2 hrs, 4° C.). The supernatant was discarded andthe pellet was resuspended in 5 ml of 3% sucrose (“DOC OMV” vaccinepreparation). The OMV and DOC OMV vaccine preparations were frozen ondry ice, and stored at −20° C. until used for immunization. The proteinconcentration of the OMV and DOC OMV preparations was determined by BCA(Pierce Chemical Co., Rockford, Ill.).

B. Immunization Schedule

MV or OMV preparations were diluted in PBS and either mixed with anequal volume of complete Freund's adjuvant (CFA; Sigma Chemical Company,St. Louis, Mo.) or aluminum hydroxide (ALHYDROGEL™ 1.3% from SuperfosBiosector, Frederikssund, Denmark), or aluminum phosphate (ALHYDROGEL™that had been incubated with PBS buffer for at least 3 hrs). In somevaccine preparations, CpG nucleotides (5′-TCCATGACGTTCCTGACGTT-3′ (SEQID NO:1) Chiron Corp., Emeryville, Calif.) were added to the aluminumphosphate/antigen mixture to a final concentration of 100 μg/ml as asecond adjuvant. Mice were immunized by the IP (CFA) or SC (aluminumphosphate, aluminum hydroxide) routes with 100 μl containing between 5to 25 micrograms of total protein of the MV prepared from themeningococcal strain RM1090.

At 3- to 4-week intervals two subsequent booster doses (5-25micrograms/mouse) were given with either incomplete Freund's adjuvant(IFA), or aluminum hydroxide, or aluminum phosphate (prepared asdescribed above) by the IP or SC routes, respectively, of first MVsprepared from meningococcal strain BZ198 and then OMVs prepared frommeningococcal strain Z1092. The sequential immunization with threedifferent meningococcal strains, which were genetically different withrespect to their serogroup, serotype, and serosubtype and other antigensconstitutes what is hereafter designated “CHORI vaccine”. In a secondexperiment, another group of mice were immunized with CHORI vaccine asdescribed above except that CpG oligonucleotides were not used as asecond adjuvant and the experiment included mice that were given theCHORI vaccine combined with aluminum hydroxide adjuvant and mice thatwere given three injections of a mixture of the MV/OMV described abovetogether with aluminum phosphate. In a third experiment, groups ofguinea pigs were given either sequential immunizations with CHORIvaccine antigens or three injections of a mixture of CHORI vaccineantigens combined with aluminum phosphate.

C. SDS-PAGE and Western Blots

Protein preparations were analyzed using 15% SDS-PAGE as described byLaemmli (1970 Nature 227: 680-685) employing a Mini-Protean IIelectrophoresis apparatus (Bio-Rad, Richmond, Calif.). Samples weresuspended in SDS sample buffer (0.06 M Tris.HCl, pH 6.8, 10% (v/v)glycerol, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10 micrograms/mlbromophenol blue) and optionally heated to 100° C. for 1 min. beforeloading directly onto the gel.

FIG. 2 shows a Coomassie-stained 15% SDS-PAGE gel of the proteinspresent in the MV of strain Z1092 (lane 2) or OMV preparations fromstrain Z1092 (lane 4), or the respective preparations after having beenextracted with 0.5% (w/v) sodium deoxycholate (i.e. DOC MV [land 3] andDOC OMV [lane 5] as described by Fredricksen et al. (1991, NIPH Ann. 14:67-79). The four corresponding preparations made from meningococcalstrains BZ198 and RM1090 ( are shown in lanes 6 to 13, respectively).Five proteins, PorA, PorB, Rmp, Opa, and Opc, are known to constitutethe major outer membrane proteins of Neisseria meningitidis. All of thepreparations appear to contain the major outer membrane proteins PorAand PorB (˜39-42 kDa) and the opacity proteins, Opa and Opc (˜28-31kDa), although the apparent mass of the particular proteins and relativeamounts were different in each preparation. The less distinct proteinhaving an apparent mass of ˜31-34 kDa in the DOC OMV preparations may bereduction modifiable protein (Rmp). In addition to these major outermembrane proteins, each MV and OMV preparation contains various otherproteins in lesser amounts. In general, the minor proteins are morevariable between strains and between MV compared to OMV preparations.

D. Antibody Binding to the Cell Surface

Binding of antibodies to the surface of live bacteria was determined byindirect fluorescence flow cytometry (Granoff et al., 1998, J. Immunol.160: 5028-5036). Bacterial cells were grown to mid-log phase inMueller-Hinton broth, harvested by centrifugation, and resuspended inblocking buffer (PBS containing 1% (w/v) bovine serum albumin (BSA) and0.4% (w/v) sodium azide) at a density of ˜10⁸ cells per ml. Dilutions oftest or control antiserum (typically 1:20, 1:200, 1:2000) were thenadded and allowed to bind to the cells, which were maintained on ice for2 hrs. Following two washes with blocking buffer, the cells wereincubated with FITC-conjugated F(ab′)2 fragment goat anti-mouse IgG(H+L) (Jackson Immune Research, West Grove, Pa.) for 1 hr. The cellswere washed twice with blocking buffer then reacted with 0.25%formaldehyde in PBS buffer before analyzing the bacterial cells by flowcytometry.

Positive control antibodies included meningococcal-specific serotypingor serosubtyping monoclonal antibodies (MN2C3B, MN16C13F4,Rijksinstituut Voor Volksgezondheid en Mileu, Bilthoven, TheNetherlands) and SEAM 12, an anti-polysaccharide monoclonal antibodythat is specific for encapsulated serogroup B strains (Granoff et al.,1998, J. Immunol. 160:5028). The negative control consisted of a mouseIgG monoclonal antibody (VIG10) of irrelevant specificity and polyclonalsera from mice immunized with membrane proteins from E. coli Antibodiesused to define serogroup, serotype, and serosubtype are provided inFIGS. 20 and 21.

FIG. 3 shows the results of a typical experiment examining antibodybinding to two test strains: MC58 and S3446. Both strains express PorAand PorB proteins that are heterologous with the respective porinprotein s from the three strains used to prepare the CHORI vaccine. Theantisera from mice immunized with CHORI antigen show an increase influorescence intensity with both strains when the antisera were testedat dilutions of 1:20 to 1:200. In contrast, polyclonal antisera preparedto proteins precipitated from culture supernatant of the E. coli showonly low intensity background fluorescence (1:20 dilution), and wereconsidered negative. Antisera from guinea pigs immunized with theNorwegian vaccine, prepared from OMV from strain H44/76 (P1.7,16), waspositive against strain MC58 with an homologous PorA serosubtype(P1.7,16) but was negative when tested at 1:20 dilution against strainS3446 with a heterologous serosubtype (P1.22, 14).

As summarized in FIG. 4, of the 12 N. meningitidis serogroup B strainstested by flow cytometry, 11 (92%), including 6 strains withheterologous PorA serosubtypes, and 3 strains having both heterologousserotypes and serosubtypes, were positive for cell surface binding byanti-CHORI antigen antisera. The one negative strain does not expressPorA, which suggests that some of the anti-CHORI vaccine antibodies bindto PorA or to proteins whose expression may be regulated in conjunctionwith PorA expression. In addition to the 11 meningococcal B strains, theanti-CHORI vaccine antisera was positive also in this assay when testedwith heterologous meningococcal serogroup A (Z1073) and C (60E) strains(FIG. 5).

E. Complement-dependent Bactericidal Antibody Activity

The bactericidal assay was adapted from the method previously describedby Mandrell et al. (1995 J. Infect. Dis. 172: 1279-1289). Finding that avaccine produces bactericidal antibodies against Neisseria meningitidisis accepted in the field as predictive of the vaccine's protectiveeffect in humans (Goldschneider et al., 1969, J. Exp. Med. 129:1307;Borrow et al. 2001 Infect Immun. 69:1568). After overnight growth onchocolate agar, several colonies were inoculated into Mueller-Hintonbroth (starting A_(620 nm) of ˜0.1) and the test organism was grown forapproximately 2 hrs. to an A_(620 nm) of ˜0.6. After washing thebacteria twice in Gey's buffer containing 1% BSA (w/v), approximately300 to 400 colony forming units (CFUs) were added to the reactionmixture. The final reaction mixture of 60 microliters contained 20%(v/v) complement, and serial 2-fold dilutions of test sera or controlmonoclonal antibodies in Gey's buffer. The complement source was humanserum from a healthy adult with no detectable anticapsular antibody toserogroup B polysaccharide when tested by ELISA (Granoff et al., 1998,J. Immunol. 160: 5028-5036), and no detectable intrinsic bactericidalactivity against the test strain at a final concentration of 20 or 40%.

In preliminary experiments with a panel of test sera, this complementsource gave comparable bactericidal titers as those obtained withagammaglobulinemic serum as the complement source. Serum bactericidaltiters were defined as the serum dilution (or antibody concentration)resulting in a 50% decrease in CFUs per ml after 60 min. incubation ofbacteria in the reaction mixture, compared to the control CFU per ml attime 0. Typically, bacteria incubated with the negative control antibodyand complement showed a 150 to 200% increase in CFU/mL during the 60min. of incubation. FIG. 6 shows data from a typical experiment withmeningococcal B strain 2996 tested with an anti-meningococcal B capsularmAb (SEAM 12, Granoff et al., 1998, J. Immunol. 160: 5028-5036), mouseand guinea pig control antisera, mouse anti-recombinant NspA andanti-CHORI antigen antisera, and guinea pig anti-Norwegian vaccineantisera.

FIG. 7 summarizes the results of measurement of complement-mediatedbactericidal activity of the anti-CHORI antigen antisera to each of themenigococcal B strains tested. All 12 strains were killed by complementtogether with similar concentrations of a positive control anti-capsularmAb (SEAM 12; subtype IgG2a (Granoff et al., 1998, J. Immunol. 160:5028-5036). Similarly, all 11 strains that were positive for anti-CHORIantisera binding by the flow assay were susceptible to antibody inducedcomplement-mediated bacteriolysis (at a 1:10 dilution or higher, eachshowed greater than 50% killing, compared to CFU/ml present at time 0).Also, the heterologous meningococcal A and C strains that were positivewhen tested in the flow assay also were positive in the bactericidalassay (FIG. 5). Again, only strain M136, which does not express PorA,and was negative for antibody binding in the flow assay, was resistantto killing in the bactericidal assay. In contrast, antibodies elicitedby either the Norwegian vaccine or rNspA, both of which aremeningococcal B vaccine candidates currently being tested in humans,were able to activate complement-mediated bacteriolysis with only alimited number of the genetically diverse set of strains. As with theflow assay, the anti-NspA vaccine antisera and the anti-Norwegianvaccine antisera were bactericidal against only a limited number of thestrains.

FIG. 8 summarizes the results of testing complement-mediatedbactericidal activity of anti-CHORI antisera prepared in a secondexperiment in mice. Data are shown for antisera prepared with CHORIvaccine given with CFA or aluminum phosphate (without CpG). The resultsare shown for the 14 meningococcal B strains tested in this experiment(8 with serosubtypes heterologous to those of the vaccine strains).Results also are shown for one additional MenB strain in which the geneencoding NspA has been inactivated (BZ198ΔNspA). All 15 strains werekilled by anti-CHORI antisera (14/15 with CHORI vaccine given with CFA;and 13/15 with CHORI vaccine given with aluminum phosphate; for theheterologous strains, 6/7 and5/7, respectively). In contrast, only 1 of15 strains was killed by antisera from control animals given 3injections of E. coli MV. These results from a second experiment in miceconfirm the earlier results obtained with the CHORI vaccine inexperiment 1. In addition, the data indicate that the second adjuvant,CpG oligonucleotides, which was not used in the second experiment, isnot needed by the CHORI vaccine to elicit broadly reactive antibody.

A group of mice in the second experiment received three injections, eachconsisting of a mixture of the same MV, MV and OMV used in thesequential immunization. The resulting antisera were bactericidalagainst 12 of the 15 strains (4 of 7 of the heterologous strains).Immunization with the mixture of antigens elicited broader bactericidalactivity than expected but the titers measured against the some strainstended to be much lower than those obtained in animals given thesequential CHORI vaccine immunization (e.g. strains CU385 and 1000,titers of 1:128 and 1:128 after CHORI vaccine/aluminum phosphate vs.titers of <1:4 and 1:6 in antisera prepared against three injections ofthe mixed antigens/aluminum phosphate).

In a third experiment, groups of guinea pigs were immunized with CHORIvaccine given with aluminum phosphate (without CpG), or aluminumhydroxide (without CpG). The results are shown in FIG. 9 for 9meningococcal B strains tested (5 with serosubtypes heterologous tothose of the vaccine strains), and for one additional MenB strain inwhich the gene encoding NspA has been inactivated (BZ198ΔNspA). 9 of the10 strains were killed by anti-CHORI antisera vs. 0 of 10 strains killedby antisera from control animals given 3 injections of E. coli MV. Thusthe CHORI vaccine elicits broad-based bactericidal antibody responses inguinea pigs, a second animal model that may be more predictive ofprotective antibody responses in humans than mice.

F. Passive Animal Protection

A criticism of bactericidal assays is that it tests the activity ofantibodies against bacteria grown in broth, and that bacteria grown invivo may have different properties. Therefore, the ability of mouseanti-CHORI antiserum to confer passive protection against N.meningitidis group B bacteremia was tested in infant rats challenged IP,using an art-accepted model and method adapted from Saukkonen et al. (J.Infect. Dis., 1988, 158: 209-212), which is regarded in the field asbeing predictive of results in humans. The meningococcal B strain 8047,which was positive by the flow cytometric assay for CHORIantigen-surface accessible epitopes and susceptible to anti-CHORIantigen bactericidal activity, was selected for this study. Infant pups(6- to 7-day old) from six litters of outbred Wistar rats (CharlesRiver, Hollister, Calif.) were randomly redistributed to the nursingmothers. Groups of five to six animals were challenged IP with 100 μl ofapproximately 5×10³ CFU of the group B strain 8047. The strain used hadbeen passaged three times in infant rats. The bacteria isolated fromblood cultures after the third pass was grown on chocolate agarovernight and stored frozen at −70° C. in vials containing sterile skimmilk. On the day of the experiment, the bacteria were grown, washed andresuspended in PBS buffer containing 1% BSA, as described above for thebactericidal assay.

The animals were given antisera or antibodies diluted in PBS containing1% BSA by IP injection 2 hrs. prior to bacterial challenge. Eighteenhours after the bacterial challenge, blood specimens were obtained bypuncturing the heart with a syringe and needle containing one to twodrops of 25 Units/ml of heparin without preservative (Fujisawa USA,Deerfield, Ill.). Aliquots of 1, 10 and 100 microliters of blood wereplated onto chocolate agar. The CFU per ml of blood was determined afterovernight incubation of the plates at 37° C. in 5% CO₂.

FIG. 10 summarizes the results of quantitative bacterial culturesperformed on blood specimens obtained 18 hrs. after challenge. A dose of10 micrograms per rat of the positive control anticapsular antibody,SEAM 3, was completely protective against the strain as was mouseanti-CHORI antisera at a dilution of 1:20. In contrast, the guinea piganti-Norwegian vaccine antisera (1:20) and the two control sera (mouseantisera prepared to -E. coli proteins or guinea pig antisera fromanimals immunized with alum alone) were not able to protect againstbacteremia caused by strain 8047.

In a second passive protection experiment using the protocol describedabove, anti-CHORI vaccine antisera prepared in guinea pigs were alsoshown to protect infant rats from meningococcal B bacteremia after IPchallenge (FIG. 11). Protection was observed in animals treated withantisera prepared to vaccine administered with aluminum phosphate oraluminum hydroxide, and was superior to the protection observed incontrol animals treated with 20 μg of a murine anticapsular monoclonalantibody (SEAM 3).

G. Immunoprecipitation of Surface Antigens Recognized by Anti-CHORIAntigen Antibodies

The method used for immunoprecipitation of surface-accessible antigenswas based on those described by Hansen et al. (1981 Infect. Immun. 33:950-953) and Gulig et al. (1982 Infect. Immun. 37: 82-88). In ourstudies, the method was used with either unlabeled cells, or with cellsin which the surface proteins had had been radioiodinated. Cells weregrown in Mueller-Hinton broth (˜7 ml) to an OD of 0.6 and harvested bycentrifugation at 5000×g at 4° C. The cell pellet was washed two timesin cold PBS containing 1% BSA or, for the radioimmunoprecipitationassay, PBS alone.

Cells to be iodinated were transferred to a glass tube. One nanomole ofKI and 1 mCi of Na¹²⁵I (Amersham,) were added to the cell suspension.Radioiodination was initiated by the addition of 0.03% H₂O₂ (50 μl) andlactoperoxidase (Sigma, St. Louis, Mo.) in water (50 μl of a 1 mg/mlsolution). The same amounts of H₂O₂ and lactoperoxidase were added at 4min. intervals for 12 min. The reaction was terminated after 16 min. byadding the reaction mixture to cold PBI (20 ml) (i.e. NaI substitutedfor NaCl in PBS). The cells were harvested by centrifugation (5,000×g,10 min, 4° C.), washed 2 times with PBS, then used immediately.

The cells were resuspended in PBS (2 ml) containing 1% BSA. The antiserawere added to 0.5 ml aliquots of cell suspension. The mixture wasincubated with rocking for 90 min. at 4° C. The cells were thencollected by centrifugation (1 min. spin in microfuge), washed two timeswith PBS/1% BSA, and resuspended in 1 ml of solubilization buffer (50 mMTris buffer, pH 7.8, containing 150 mM NaCl, 1 mM EDTA, 1% Triton X-100,0.2% sodium deoxycholate, and 0.1% sodium dodecyl sulfate. Afterincubation for 60 min. at 37° C., the insoluble material was removed bycentrifugation (45,000×g for 60 min. at 20° C.). The supernatants werethen transferred to tubes containing 3-4 milligrams of protein ASepharose beads (Sigma) pre-equilibrated with 50 μl of PBS. The sampleswere incubated overnight at 4° C. with rocking. The beads were washedfive times with solubilization buffer. Bound proteins were released fromthe beads by adding 75 μl of SDS sample buffer and heated to 100° C. for1 min. After removing the supernatant, 1 μl of 2-mercapto ethanol wasadded to each sample and then heated again to 100° C. for 1 min. Thesamples were then run on a 15% SDS-PAGE gel and stained using silverstain (Pierce Chemical Co., Rockford, Ill.).

For Western blots, the gel was equilibrated with buffer (48 mM Tris.HCl,39 mM glycine, pH 9.0, 20% (v/v) methanol) and the proteins weretransferred to a nitrocellulose membrane (Bio-Rad) using a Trans-Blot™(Bio-Rad) semi-dry electrophoretic transfer cell. The nitrocellulosemembranes were blocked with 2% (w/v) skim milk in PBS containing 0.2%(w/v) sodium azide. Antisera were diluted in the same blocking buffercontaining 0.1% Tween-20. Bound antibody was detected using rabbitanti-mouse IgG, A, M-alkaline phosphatase conjugate polyclonal antibody(Zymed, South San Francisco, Calif.) diluted in PBS containing 1% (w/v)BSA, 1% (w/v) Tween-20, and 0.2% (w/v) sodium azide and Sigma Fast™BCIP/NBT substrate (Sigma).

FIG. 12 is a silver-stained SDS gel demonstrating cell surface proteinsprecipitated with the anti-CHORI antisera (Lane 2) from M7, anon-encapsulated mutant of serogroup B strain, NMB (Stephens et al.1991, Infect. Immun. 59: 4097-4107), six proteins having apparent massesof 59.5, 40.7. 39.6, 33, 27.9, and 14.5 kDa were precipitated by theantisera prepared to the CHORI antigen (Lane 2) but not by controlanti-E. coli protein antisera (Lane 3). The same results were obtainedwhen the anti-CHORI antigen antisera were used to precipitate surfaceproteins from the encapsulated parent strain, NMB (data not shown).Except for the 59.5 and 27.9 kDa heavy and light chain Ig proteins (seebelow), the same surface proteins detected by silver staining wereobserved also in ¹²⁵I-labeled cells (data not shown).

Of note, there was no lipooligosaccharide (LOS), which would be detectedby silver staining, that was precipitated by the anti-CHORI antigenantisera. Further experiments, described below, were designed todetermine whether the observed surface binding and protective biologicalactivity of anti-CHORI vaccine antisera were due to anti-LOS antibodies.

FIG. 13 shows a Western blot of the same samples as resolved on the SDSgel in FIG. 12. Samples in lanes 1 to 3 were detected by anti-CHORIvaccine antisera. Samples in Lanes 4 to 6 were detected by an anti-PorAP1.2-specific mAb. The proteins having apparent masses of 59.5 and 27.9kDa in FIG. 13, (Lanes 2, 3, 5, and 6) correspond to antibody heavy andlight chains, respectively, as they were detected with the rabbitanti-mouse Ig alkaline phosphatase conjugate secondary antibody. The40.7 kDa protein precipitated by the anti-CHORI antigen antisera (FIG.12, Lane 2) was reactive with the anti-PorA P1.2-specific antibody inthe Western (FIG. 13, Lane 5) and is, therefore, PorA P1.2. As expected,PorA P1.2 also was detected in the total protein from M7 (FIG. 13, Lane4). The anti-CHORI vaccine antisera detected only the 33 kDa protein(Lane 2) and not PorA. Thus, the antisera prepared to the CHORI antigenreacts with both native and denatured 33 kDa protein but only nativeforms of the 40.7. 39.6, and 14.5 kDa proteins present on the cellsurface of strain M7 (FIG. 12, lane 2).

Similar immunoprecipitation experiments were performed on the MV and OMVpreparations used for immunization and seven genetically diverse,encapsulated serogroup B strains. The results are summarized in FIG. 14.When the results are compared, it is apparent that the sequentialimmunization with membrane vesicles from three genetically diversemeningococcal strains elicits antibodies that recognize a variety ofantigens. Some of the antigens that are recognized are the same in allstrains; others are strain specific or common to subsets of strains. Forexample, proteins having apparent masses of 37-41 kDa were precipitatedfrom strains BZ198 and NMB but not from any of the other strains.Similarly, proteins having an apparent mass of 25.7 kDa wereprecipitated from strains NG3/88 and S3446 but not from any otherstrains. However, there is one surface protein having an apparent massof 32 to 33 kDa that is recognized by anti-CHORI antigen antisera in allof the examples except for strain M136, which was negative in both theflow and bactericidal assays. The 32 to 33 kDa protein may be aconserved antigen.

Additional experiments were performed on encapsulated serogroup Bstrains. CU385, BZ198 and 1000 using cells in which the surface proteinshad had been radioiodinated (FIG. 14A) and precipitated with immuneantisera from different groups of mice given the CHORI vaccine. Inaddition to the proteins described above, in this second set ofexperiments, proteins with apparent molecular masses between 10 and 14.5kDa, and 80 kDa were precipitated from strains CU385 and BZ198. Threeproteins with apparent kDa of 26, 41 and 45 were precipitated fromstrain 1000.

It is important to note that not all antigens recognized by the antiserafrom mice immunized with CHORI antigen were immunoprecipitated from thebacterial cells. There were also antibodies in the antisera to someantigens (e.g. the NspA protein) that were shown to be present by anELISA or Western blot, but were not immunoprecipitated in thisexperiment. The failure to detect these other antigens may result fromthe fact that the antibody/antigen complex must be stable in thepresence of detergents (Triton X-100, deoxycholate, and lauryl sulfate)to be detected.

H. Detection of Proteins Reactive in CHORI Vaccine MV and OMVPreparations with Anti-CHORI Vaccine Antibodies Elicited in Mice andGuinea Pigs.

FIG. 15 shows a Western blot of MVs from strains RM1090(C:2a:P1.5,2:L3,7), and BZ198 (B:NT:P1.4) and OMV from strain Z1092(A:4:P1.10). Antisera from mice immunized CHORI vaccine combined withCFA, or given with aluminum phosphate adjuvant, or from guinea pigsimmunized with CHORI vaccine together with aluminum phosphate adjuvant,were used as the primary detecting antibody. The blot shows that theCHORI vaccine elicits antibodies that are reactive with several proteinshaving similar apparent molecular mass in each of the MV or OMVpreparations independent of the animal species or adjuvant used in thevaccine. The apparent molecular masses of all proteins in the MV or OMVpreparations that are reactive with antibodies produced by immunizationwith CHORI vaccine are summarized in FIG. 16.

I. Detection of Anti-LOS Antibody Activity

One of the antigenic determinants on the surface of the meningococcithat has been observed to elicit bactericidal antibodies islipooligosaccharide (LOS). In order to determine whether anti-LOSantibodies were elicited by the CHORI vaccine, a LOS affinity column wasprepared and used to absorb out anti-LOS antibodies in the anti-CHORIvaccine antisera using methods described by Shenep et al. (1982, J.Infect. Dis. 145: 181-190) with the following modifications. LOS wasprepared from each vaccine strain by the method of Appicella et al.(Bacterial Pathogensis (1997) V. L. Clark and P. M. Bavoil eds. AcademicPress, San Diego, Calif.), and was conjugated to BSA as follows. LOS (1mg) was combined with BSA (2 mg) in 100 mM MES buffer, pH 5.0. EDC(1-ethyl-3-(3-dimehtylaminopropyl)carbodiimide HCl; 100 μl of a 10 mg/mlsolution in water) was added with stirring followed by incubation atambient temperature for 2 hrs. An equal mixture of the three LOS-BSAconjugates (1 mg LOS-BSA conjugate per ml of hydrated gel) was coupledto CNBr-activated agarose beads (Sigma Chemical Co., St. Louis, Mo.) insodium carbonate buffer (0.1 M, pH 8.0) overnight at ambienttemperature.

After removal of anti-LOS antibodies by passing the anti-CHORI vaccineand anti-CHORI mixed antigen antisera through the LOS affinity column,the antisera were concentrated to their original volume byultrafiltration and tested for the presence of anti-LOS antibody byELISA and complement-mediated bactericidal activity against two MenBstrains (BZ198 and S3032). As summarized in FIG. 17, the presence ofanti-LOS antibody was greatly reduced or eliminated by absorption withthe LOS-BSA conjugate affinity column. As shown in FIG. 18, there waslittle or no difference in the bactericidal titers between the absorbedand unabsorbed sera from mice or guinea pigs immunized with CHORIvaccine indicating that anti-LOS antibody does not contributesignificantly to the bactericidal activity against either a vaccinestrain (BZ198) or strain S3032, which has PorA and PorB that areheterologous to the strains used to prepare the vaccine. There was agreater effect on the bactericidal titers of antisera prepared from miceand guinea pigs immunized with Mixed CHORI vaccine indicating a moresignificant contribution of anti-LOS antibodies to the bactericidaltiter in this antisera.

J. Preparation of Monoclonal Antibodies.

Female CD1 mice (Charles River, Hollister, Calif.) were vaccinatedsequentially with MV prepared from meningococcal strain RM1090(C:2a:P1.5,2), BZ198 (B:NT:7,4), and OMV from strain Z1092(A:4,21:P1.10). The mice were given three 100 microliters injections,each separated by three weeks, containing 5 micrograms of protein. Thefirst two doses were given subcutaneously together with aluminumphosphate (0.5% wt/vol) and the final dose was given without adjuvantand administered intraperitoneally (i.p.). Three days later, the animalswere sacrificed and their spleen cells were fused with myeloma cells(P3X63-AG8.653) at a ratio of 1 spleen cell to 1.7 myeloma cell. Aftertwo weeks incubation in HAT selective medium, hybridoma supernatantswere screened for antibody binding activity by whole cell ELISA usingencapsulated MenB strains 1000 and CU385 as the target antigen. Themethod described by Abdillahi and Poolman, (Microb Pathog. 1988 4:27-32)was used for the whole cell ELISA assay. Hybridomas secreting antibodythat was reactive with both 1000 and CU385 strains in a whole cell ELISAand were positive for binding by flow cytometry were cloned by limitingdilution and then expanded and frozen for subsequent use in tissueculture.

Antibodies from eight cell lines were characterized in detail. Thesubclasses of the monoclonal antibodies were determined using anantibody capture ELISA and alkaline phosphatase-conjugated polyclonalantibody specific for each of the mouse IgG subclasses, IgM, IgA, and κand λ light chains (Southern Biotechnology Associates, Inc. Birmingham,Ala.). The monoclonal antibodies produced by the hybridoma clones wereharvested from tissue culture media by ammonium sulfate precipitation(55% wt/vol). The concentration of the purified mAb was determined bycapture ELISA using Ig standards as recommended by the manufacturer(Southern Biotechnology Associates, Inc. Birmingham, Ala.).

K. Reactivity of Anti-CHORI Antigen mAbs with Diverse MenB Strains.

The ability of mAbs prepared from mice immunized with anti-CHORI antigen(administered with CFA or aluminum phosphate) to bind to diverse MenBstrains was determined by whole cell ELISA (Abdillahi and Poolman,Microb Pathog. 1988 4:27-32). The results are summarized in FIG. 19.None of the monoclonal antibodies react with LOS prepared from theimmunizing strains. The mAbs 1D9 and 14C7 are reactive with antigens inall or nearly all meningococcal strains tested but not with anynon-neisserial strains. The mAb 14C7 is specific for the highlyconserved Neisserial surface protein NspA since it is reactive withrNspA expressed in E. coli. This mAb also is reactive with strains 8047and BZ198 but is not reactive with the corresponding strains in whichthe NspA gene has been inactivated. In contrast to the broadly reactiveantibodies, antigens recognized by the mAbs 4B11 and 9B6 are limited tocertain strains. Note that MAb 4B11 is reactive with strain 8047 but notwith the corresponding 8047 mutant in which the NspA gene has beeninactivated. However, mAb 4B11 does not bind to rNspA expressed in E.coli vesicles, and also does not bind to strain BZ198, which is known tonaturally overexpress (see Moe et al. (1999 Infect. Immun. 67: 5664; Moeet al. Infect Immun. 2001 69:3762). Therefore, mAb 4B11 may recognize aNspA epitope that is specific to strain 8047, or an epitope on anothermembrane protein that is not present in strain BZ198 but is present andassociated with NspA expression in strain 8047. Taken together, theresults with the different mAbs show that the anti-CHORI antigen vaccineelicits antibodies against both highly conserved and non-conservedproteins.

L. Bactericidal Activity of Anti-CHORI Antigen mAbs with Diverse MenBStrains.

The complement-mediated bactericidal activity of mAbs prepared from miceimmunized with anti-CHORI antigen and tested against several MenBstrains is summarized in FIG. 20. The monoclonal antibody 1D9, whichreacts by ELISA with all N. meningitides strains tested, was notbactericidal. The monoclonal antibody 14C7, which appears to recognizeNspA, was bactericidal or bacteriostatic against all strains testedexcept BZ198ΔNspA (a knockout of NspA). The activity of the 14C7monoclonal antibody was superior to that of the control monoclonalantibody AL12 (produced in mice immunized with recombinant NspA (see Moeet al. Infect Immun. 2001 69:3762). This observation suggests thatimmunization with the CHORI vaccine provides a superior means foreliciting bactericidal anti-NspA antibodies as compared to immunizationwith a recombinant NspA-based vaccine.

The monoclonal antibody 4B11 (an IgM antibody) was bactericidal againststrains 1000 and CU385. Note that the 4B11 monoclonal antibody did notreact with these same strains by whole-cell ELISA at the highestconcentration tested (see FIG. 19). The bactericidal assays measuresfunctional antibody activity using live bacteria whereas the bacterialcell ELISA measures antibody binding to heat-killed bacteria, which inthese strains may have denatured the antigenic target of mAb 4B11.

DEPOSITS

A deposit of biologically pure cultures of the materials in the tablebelow was made with the American Type Culture Collection, 10801University Blvd., Manasas, Va. 20110-2209, under the provisions of theBudapest Treaty, on or before the filing date of the presentapplication. The accession number indicated is assigned after successfulviability testing, and the requisite fees were paid. Access to saidcultures will be available during pendency of the patent application toone determined by the Commissioner to be entitled to such under 37C.F.R. §1.14 and 35 U.S.C. §122. All restriction on availability of saidcultures to the public will be irrevocably removed upon the granting ofa patent based upon the application. Moreover, the designated depositswill be maintained for a period of thirty (30) years from the date ofdeposit, or for five (5) years after the last request for the deposit;or for the enforceable life of the U.S. patent, whichever is longer.Should a culture become nonviable or be inadvertently destroyed, or, inthe case of plasmid-containing strains, lose its plasmid, it will bereplaced with a viable culture(s) of the same taxonomic description.

These deposits are provided merely as a convenience to those of skill inthe art, and are not an admission that a deposit is required. A licensemay be required to make, use, or sell the deposited materials, and nosuch license is hereby granted. The deposit below was received by theATCC on or before the filing date of the present application.

Description ATCC Accession No. Hybridoma 1D9 XX1 Hybridoma 4B11 XX2Hybridoma 9B8 XX3 Hybridoma 14C7 XX4 MenC strain RM1090 XX5 MenB strainBZ198 XX6 MenA strain 71092 XX7

1. A method of eliciting broad spectrum protective immunity againstNeisseria meningitidis, said method comprising the steps of:administering to a mammal a first preparation of microvesicles (MVs)from a first Neisseria meningitidis species that is a member of a firstserotype and of a first serosubtype, in an amount sufficient to elicitan immune response to epitopes present in said first preparation; andadministering to said mammal a second preparation of MVs from a secondNeisseria meningitidis species that is a member of a second serotype andof a second serosubtype, in an amount sufficient to elicit an immuneresponse to epitopes present in said second preparation; wherein theserotype or serosubtype of each of the first and second Neisseriameningitidis species is different, and wherein administering of thefirst and second preparations is sufficient to elicit an immune responsein said mammal, wherein said immune response confers protective immunityagainst more than one strain of Neisseria meningitidis species.
 2. Themethod of claim 1, the method further comprising: administering to saidmammal a third preparation of outer membrane vesicles (OMV), MVs, orboth OMVs and MVs from a third Neisseria meningitidis species that is amember of a third serotype and of a third serosubtype, in an amountsufficient to elicit an immune response to epitopes present in saidthird preparation.
 3. The method of claim 2, wherein the first, second,and third preparations are administered serially.
 4. The method of claim3, wherein the preparations are administered such that the firstpreparation is administered first, the second preparation administeredsecond, and third preparation administered third.
 5. The method of claim1, wherein the first and second preparations are administered as amixture.
 6. The method of claim 2, wherein the third preparationcomprises MVs.
 7. The method of claim 1, wherein protective immunity isconferred against at least four strains of Neisseria meningitidisspecies.
 8. The method of claim 7, wherein protective immunity conferredis against more than one strain of serogroup B Neisseria meningitidisspecies.
 9. The method of claim 1, wherein the OMV and MV preparationsare administered together with pharmaceutically acceptable excipients.10. The method of claim 9, wherein the excipients comprise an adjuvant.11. The method of claim 10, wherein the adjuvant is aluminum phosphate,aluminum hydroxide, alum or MF59.
 12. The method of claim 1, whereinadministering is by injection.
 13. The method of claim 1, whereinadministering is oral or by aerosol administration.
 14. The method ofclaim 1, wherein the mammal is a human.
 15. The method of claim 14,wherein the human is immunologically naive with respect to Neisseriameningitidis.
 16. The method of claim 14, wherein the human is a humanchild less than five years old.
 17. The method of claim 1, wherein thefirst and second preparations are treated to reduce endotoxin.
 18. Themethod of claim 17, wherein endotoxin reduction is by detergentextraction with a detergent other than deoxycholate.
 19. A method ofeliciting broad spectrum protective immunity against Neisseriameningitidis, said method comprising the steps of: administering to amammal a first preparation of microvesicles (MVs) from a first Neisseriameningitidis species that is a member of a first serosubtype, in aamount sufficient to elicit an immune response to epitopes present insaid first preparation; administering to said ruanimal a secondpreparation of MVs from a second Neisseria meningitidis species that isa member of a second serosubtype, in a amount sufficient to elicit animmune response to epitopes present in said second preparation; whereinthe serosubtype of each of the first and second Neisseria meningitidisspecies is different, and wherein administering of the first and secondpreparations is sufficient to elicit an immune response in said mammal,wherein said immune response confers protective immunity against atleast four strains of Neisseria meningitidis species.
 20. The method ofclaim 19, wherein the method further comprises administering to saidmammal a third preparation from a third Neisseria meningitidis speciesthat is a member of a third serosubtype, the third preparationcomprising outer membrane vesicles (OMV), MVs, or both OMVs and MVs,said administering being in an amount sufficient to elicit an immuneresponse to epitopes present in said third preparation, wherein theserosubtype of the first and third species is different.
 21. The methodof claim 19, wherein the first and second preparations are administeredas a mixture.
 22. The method of claim 19, wherein the first and secondpreparations are administered serially.
 23. The method of claim 19,wherein the first and second preparations are treated to reduceendotoxin.
 24. The method of claim 23, wherein endotoxin reduction is bydetergent extraction with a detergent other than deoxycholate.
 25. Amethod of eliciting broad spectrum protective immunity against aNeisseria meningitidis species, said method comprising the steps of:administering to a mammal a first preparation from a first Neisseriameningitidis species, the first preparation comprising outer membranevesicles (OMV), microvesicles (MV), or both OMV and MV, saidadministering of the first preparation being in an amount sufficient toelicit an immune response to epitopes present in said first preparation;administering to the mammal a second preparation from a second Neisseriameningitidis species that is genetically diverse to the first Neisseriameningitidis species, the second preparation comprising outer membranevesicles (OMV), microvesicles (MV), or both OMV and MV, saidadministering of the second preparation being in an amount sufficient toelicit an immune response to epitopes present in said secondpreparation; wherein administering of the first and second preparationselicits an immune response in said mammal, wherein said immune responseconfers protective immunity against more than one strain of Neisseriameningitidis species.
 26. The method of claim 25, comprising theadditional step of administering to said mammal a third preparation ofouter membrane vesicles from a third Neisseria meningitidis species,which third species that is genetically diverse to at least the firstNeisseria meningitidis species, said administering being in an amountsufficient amount to elicit an immune response to epitopes present insaid third preparation.
 27. The method of claim 25, wherein the firstand second preparations are administered serially.
 28. The method ofclaim 25, wherein the first and second preparations are treated toreduce endotoxin.
 29. The method of claim 28, wherein endotoxinreduction is by detergent extraction with a detergent other thandeoxycholate.
 30. A method of eliciting broad spectrum protectiveimmunity against a Neisseria meningitidis species, said methodcomprising the steps of: administering to a mammal a first preparationfrom a first Neisseria meningitidis species that is a member of a firstserotype and of a first serosubtype, the first preparation comprisingouter membrane vesicles (OMV), microvesicles (MV), or both OMV and MV,said administering of the first preparation being in an amountsufficient to elicit an immune response to epitopes present in saidfirst preparation; administering to the mammal a second preparation froma second Neisseria meningitidis species that is a member of a secondserotype and of a second serosubtype, the second preparation comprisingouter membrane vesicles (OMV), microvesicles (MV), or both OMV and MV,said administering of the second preparation being in an amountsufficient to elicit an immune response to epitopes present in saidsecond preparation; wherein the serotype or serosubtype of each of thefirst and second Neisseria meningitidis species is different, andwherein administering of the first and second preparations elicits animmune response in said mammal, wherein said immune response confersprotective immunity against more than one strain of Neisseriameningitidis species.
 31. The method of claim 30, further comprising:administering to said mammal a third preparation of outer membranevesicles (OMV), MVs, or both OMVs and MVs from a third Neisseriameningitidis species that is a member of a third serotype and of a thirdserosubtype, in an amount sufficient to elicit an immune response toepitopes present in said third preparation.
 32. The method of claim 31,wherein the first, second, and third preparations are administeredserially.